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Abstract Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)‐to‐mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b‐expressing cells by using novel Wnt2b‐2A‐CreERT2 knock‐in and LacZ‐reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b‐expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b^{2A‐CreERT2/+};R26R^LacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b‐expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ⁺ epicardium‐derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b‐expressing PEO and epicardial cells to the postnatal heart.

The dorsolateral striatum (DLS) of rodents is functionally subdivided into somatotopic subregions that represent each body part along both the dorsoventral and anteroposterior (A-P) axes and play crucial roles in sensorimotor functions via corticostriatal pathways. However, little is known about the spatial gene expression patterns and heterogeneity of spiny projection neurons (SPNs) within somatotopic subregions. Here, we show that the cell adhesion molecule gene Cdh20, which encodes a Type II cadherin, is expressed in discrete subregions covering the inner orofacial area and part of the forelimb area in the ventral domain of the DLS (v-DLS) in rats. Cdh20-expressing cells were localized in the v-DLS at the intermediate level of the striatum along the A-P axis and could be classified as direct-pathway SPNs or indirect-pathway SPNs. Unexpectedly, comprehensive analysis revealed that Cdh20 is expressed in SPNs in the rat DLS but not in the mouse DLS or the ferret putamen (Pu). Our observations reveal that Cdh20 expression demarcates somatotopic subregions and subpopulations of SPNs specifically in the rat DLS and suggest divergent regulation of genes differentially expressed in the v-DLS and Pu among mammals.

BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme. This article is protected by copyright. All rights reserved.

Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4-/-;Six5-/- fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4-/-;Six5-/- embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4-/-;Six5-/- embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4-/-;Six5-/- embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.

The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.

This unit describes basic methods formammalianwhole embryo culture (WEC) using embryonic day 10.5 mouse embryos, including the preparation of highquality immediately centrifuged (IC) rat serum that is commonly used for WEC and is essential for normal growth and development of cultured mouse and rat embryos in vitro. An alternative protocol for different stages of rodent embryos is also introduced. Since embryos for WEC are dissected out of the uterus and manipulated under the microscope, one can overcome many of the difficulties of gene delivery encountered using in utero electroporation. A description for a gene transfer method to label neural stem/progenitor cells of the cortical primordium in a highly region-specific manner is also included.

The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-20-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals.

The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO(2)/pH and pO(2) levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Botzinger complex group (preBotC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O-2 sensor molecules in the adult central and peripheral nervous systems.

Mammalian whole embryo culture ( WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged ( IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos ( i. e., E8.0- 12.5 for the mouse, and E10.0- 14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Pax6 balances cell proliferation and neuronal differentiation in the mammalian developing neocortex by regulating the expression of target genes. Using microarray analysis, we observed the down-regulation of Dmrta1 (doublesex and mab-3-related transcription factor-like family A1) in the telencephalon of Pax6 homozygous mutant rats (rSey(2)/rSey(2)). Dmrta1 expression was restricted to the neural stem/progenitor cells of the dorsal telencephalon. Overexpression of Dmrta1 induced the expression of the proneural gene Neurogenin2 (Neurog2) and conversely repressed Ascl1 (Mash1), a proneural gene expressed in the ventral telencephalon. We found that another Dmrt family molecule, Dmrt3, induced Neurog2 expression in the dorsal telencephalon. Our novel findings suggest that dual regulation of proneural genes mediated by Pax6 and Dmrt family members is crucial for cortical neurogenesis.

The mammalian cerebral cortex develops from proliferative apical progenitor cells (APs) that exhibit cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM), which may be important for efficient and continuous production of neurons. The Pax6 transcription factor plays a major role in INM by regulating various downstream molecules. We have previously observed abnormal INM and unstable localization of the centrosome in APs of the Pax6 homozygous mutant rat embryo. To understand the mechanisms of INM, we focused on the centrosomes of APs. One of the centrosomal proteins, ninein, is specifically localized in the centrosome of APs. We observed a dramatic downregulation of ninein in APs of the Pax6 mutant. Moreover, knockdown of ninein by RNAi induced ectopic distribution of reduced numbers of BrdU-positive (S-phase) and PH3-positive (M-phase) cells. Furthermore, time-lapsed imaging demonstrated that knockdown of ninein in vivo induced abnormal INM. Finally, we observed impaired microtubule regrowth in neural progenitors taken from Pax6 homozygous mutant rat embryos, which was recovered by via ninein overexpression. We also found that ninein knockdown enlarged the surface size area of apical endfeet of the APs. Our results suggest that ninein plays a role in the molecular machinery essential for INM by connecting microtubules to the centrosome. (C) 2013. Published by The Company of Biologists Ltd.

Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest-derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFR beta and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC-derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2-negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC-derived pericytes may be still in pre-mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon.

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved. The EMBO Journal (2012) 31, 1879-1892. doi:10.1038/emboj.2012.43; Published online 6 March 2012 Subject Categories: cell & tissue architecture; neuroscience

The vertebrate neuroepithelium can be subdivided into non-boundary and boundary regions. The boundary regions act as signaling centers for regional specification and neurogenesis in the neighboring non-boundary regions. An important question is how boundary regions are specified and maintained during brain development. In this study, we report that Pax6, a homeodomain transcription factor, regulates boundary-cell specification between rhombomeres of the developing rat hindbrain. We compared expression patterns of four boundary-cell markers, including PLZF (Zbtb16), Ring1A (Ring1), Wnt5a, and cadherin7 (Cdh7), in wild-type and Pax6 loss-of-function mutant hindbrains and found that the expression of Zbtb16, Ring1, and Wnt5a was down-regulated in the rhombomere boundaries, while Cdh7 expression was up-regulated in the non-boundary regions of the Pax6 mutant hindbrain. Morphological observations revealed that the boundary regions were larger and that the interface between the boundary and non-boundary regions was obscured in the Pax6 mutant hindbrain. We also found ectopic neuronal differentiation in the boundary cells of the Pax6 mutant hindbrain. In addition, we observed that Hes5 was down-regulated and that Neurogenin2 (Neurog2) was up-regulated in the boundary regions of the Pax6 mutant hindbrain. Because Hes genes have been shown to inhibit neuronal differentiation by repressing proneural genes, Pax6 may act through this pathway to prevent neurogenesis in the boundary cells. Taken together, Pax6 seems to be required for coordinating boundary-cell specification and reducing neurogenesis within the hindbrain boundary region. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Cranial motor neurons, which are divided into somatic motor (SM), branchiomotor (BM) and visceral motor (VM) neurons, form distinct axonal trajectories to innervate their synapse targets. Rho GTPase regulates various neuronal functions through one of the major effector proteins, Rho-kinase. Here, we addressed the in vivo role of the Rho/Rho-kinase signaling pathway in axon patterning of cranial motor neurons. We performed conditional expression of a dominant-negative mutant for RhoA or Rho-kinase in transgenic mice by using the Cre-loxP system to suppress the activity of these molecules in developing cranial motor neurons. Blockade of the Rho/Rho-kinase signaling pathway caused defects in the patterning of SM axons but not in that of BM/VM axons, in which defects were accompanied by reduced muscle innervation and reduced synapse formation by SM neurons. In addition, blockade of the signaling pathway shifted the trajectory of growing SM axons in explant cultures, whereas it did not appear to affect the rate of spontaneous axonal outgrowth. These results indicate that the Rho/Rho-kinase signaling pathway plays an essential role in the axon patterning of cranial SM neurons during development.

Background: The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays. Results: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up-or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally. Conclusions: These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

Six family transcription factor genes play multiple and crucial roles in the development of the vertebrate sensory system including the eye, olfactory epithelium and otic vesicle, and these genes are highly expressed in the neural crest-derived cranial mesenchymal cells in the mouse embryo. However, expression patterns have yet to be determined for the Six family genes in the developing tooth germ. In this study, we examined expression of six members of the Six family genes in the dental mesenchyme and the dental epithelium of the developing tooth germs in mice by in situ hybridization. We found dynamic expression patterns for Six1, Six2, Six4 and Six5 in the oral epithelium and mesenchymal cells with distinct expression patterns at the early stage before invagination of the dental epithelium. In addition, expression of Six1 and Six4 was observed in the inner enamel epithelium of the incisor and molar tooth germs at the cap stage. Expression of Six5 was maintained in the bell stage tooth germs, and intense expression of Six1 and Six4 was detected not only in the mesenchyme-derived dental follicle but also in the proliferating inner enamel epithelium of the labial cervical loop of the incisor tooth germ. Taken together, our results suggest that dynamic expression of Six family genes represents specific stages of the developing tooth germ. This dynamic expression is embodied in changes in both space and over time, and these changes in expression suggest that Six family genes may participate in tooth germ morphogenesis and the proliferation and/or differentiation of the incisor ameloblast stem/progenitor cells.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft, and introduce various applications of WEC technique for studies in mammalian developmental biology. © JoVE 2006-2011 All Rights Reserved.

Background: Vertebrate classic cadherins are divided into type I and type II subtypes, which are individually expressed in brain subdivisions (e.g., prosomeres, rhombomeres, and progenitor domains) and in specific neuronal circuits in region-specific manners. We reported previously the expression of cadherin19 (cad19) in Schwann cell precursors. Cad19 is a type II classic cadherin closely clustered on a chromosome with cad7 and cad20. The expression patterns of cad7 and cad20 have been reported previously in chick embryo but not in the developing and adult central nervous system of mammals. In this study, we identified rat cad7 and cad20 and analyzed their expression patterns in embryonic and adult rat brains. Results: Rat cad7 protein showed 92% similarity to chick cad7, while rat cad20 protein had 76% similarity to Xenopus F-cadherin. Rat cad7 mRNA was initially expressed in the anterior neural plate including presumptive forebrain and midbrain regions, and then accumulated in cells of the dorsal neural tube and in rhombomere boundary cells of the hindbrain. Expression of rat cad20 mRNA was specifically localized in the anterior neural region and rhombomere 2 in the early neural plate, and later in longitudinally defined ventral cells of the hindbrain. The expression boundaries of cad7 and cad20 corresponded to those of region-specific transcription factors such as Six3, Irx3 and Otx2 in the neural plate, and Dbx2 and Gsh1 in the hindbrain. At later stages, the expression of cad7 and cad20 disappeared from neuroepithelial cells in the hindbrain, and was almost restricted to postmitotic cells, e. g. somatic motor neurons and precerebellar neurons. These results emphasized the diversity of cad7 and cad20 expression patterns in different vertebrate species, i.e. birds and rodents. Conclusion: Taken together, our findings suggest that the expression of cad7 and cad20 demarcates the compartments, boundaries, progenitor domains, specific nuclei and specific neural circuits during mammalian brain development.

Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.

The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex.

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.

The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of the new kidney.

To identify a novel type II classical cadherin, we searched the genome database and found rat cadherin19 (cad19) with high similarity to human cadherin19. We also found nucleotide sequences corresponding to rat cad19 in mouse and chicken genomes. In situ hybridization of rat cad19 revealed that rat cad19 mRNA was initially expressed in cephalic neural crest cells, and then in the cranial ganglia, migrating trunk neural crest cells, the nascent dorsal root ganglia, and the sympathetic ganglia. Expression of cad19 overlapped with that of neural crest markers, including Sox10 and AP-2, but cad19 expression was confined to subpopulations of the neural crest-derived cells, those typically observed in the satellite glia at the periphery of the ganglia and Schwann cell precursors along the peripheral nerves. cad19 mRNA was not detected in cells expressing Phox2b, an epibranchial placode-derived neurons, nor in those expressing neuronal markers such as Hu protein. These observations suggest that cad19 is expressed in neural crest-derived, non-neuronal cells. Although the expression of cad19 mRNA persisted in Schwann cell precursors at E14.5, it was no longer detected in maturing Schwann cells at later stages. These results suggest that cad19 is an evolutionarily conserved cadherin and may be involved in the early development of Schwann cells in the peripheral nervous system. (C) 2004 Wiley-Liss, Inc.

The central nervous system of the mammalian embryo is organized according to the expression of region-specific transcription factors along the anteroposterior and/or the dorsoventral axis. For example, the dorsal ventricular zone (VZ) of the embryonic spinal cord expresses Pax3 and Pax7, the ventral VZ expresses Pax6, and the more ventral VZ expresses Nkx2.2. Properties of neuronal precursors located in the VZ are determined by the characteristic expression patterns of these transcription factors, leading to the generation of distinct classes of neurons. Recent studies demonstrated that radial glial cells produce neurons in addition to glia during central nervous system development. Thus, neuronal precursor diversity may be dependent upon the diversity of radial glial cells. To investigate this hypothesis, we analyzed the expression of radial glial cell markers and transcription factors in the mouse embryonic spinal cord. We show that radial glial cells indeed express domain-specific transcription factor. Moreover, they varied in expression of the astrocyte-specific glutamate transporter. The region where the astrocyte-specific glutamate transporter is strongly expressed in the ventral radial glial cells is closely related to the Pax6-expressing domain, and the weakly expressing region corresponding to the Nkx2.2-expressing domain. Furthermore, dorsal radial fibers expressed ephrin-B1. Thus, different types of radial glial cells exist in different domains defined by the transcription factor expression at E12.5. We also show that this diversity continues to the gliogenic stage of radial glial cells. This raises the idea that astrocytes generated from different domains along the dorsoventral axis in the mouse spinal cord have distinct characteristics. Copyright (c) 2005 S. Karger AG, Basel.

A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPaseRho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine-threonine protein kinase (Rho-kinase-ROK-ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA(RhoADN) or for Rho-kinase (Rho-KDN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DNshowed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.

Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Galpha subunits, Galphai has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.

One of the goals of developmental neuroscience in the post-genomic era is to clarify functions of a huge number of anonymous genes of which only DNA sequences are identified. More convenient methods for genetic manipulation in vertebrates, especially mammals, could help us to identify functions of the novel genes. Here we introduce a novel gene transfer technology using electroporation (EP), which is a simple and powerful strategy for genetic analysis. We have applied this method to cultured mammalian embryos in order to understand the function of specific genes in the developing brain. We have also performed EP in developing fetuses in utero guided by ultrasound image. The combination of these techniques in addition to analysis of genetic mutants will clarify functions of individual genes, gene interactions, and the molecular mechanisms underlying the brain development.

Recent studies have shown that generation of different kinds of neurones is controlled by combinatorial actions of homeodomain (HD) proteins expressed in the neuronal progenitors. Pax6 is a HD protein that has previously been shown to be involved in the differentiation of the hindbrain somatic (SM) motoneurones and V1 interneurones in the hindbrain and/or spinal cord. To investigate in greater depth the role of Pax6 in generation of the ventral neurones, we first examined the expression patterns of HD protein genes and subtype-specific neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. We found that Islet2 (SM neurone marker) and En1 (V1 interneurone marker) were transiently expressed in a small number of cells, indicating that Pax6 is not directly required for specification of these neurones. We also observed that domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbxl) were shifted and their boundaries became blurred. Thus, Pax6 is required for establishment of the progenitor domains of the ventral neurones. Next, we performed Pax6 overexpression experiments by electroporating rat embryos in whole embryo culture. Pax6 overexpression in the wild type decreased expression of Nkx2.2, but ectopically increased expression of Irx3, Dbxl and Dbx2. Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescued the development of Islet2-positive and En1-positive neurones. To know reasons for perturbed progenitor domain formation in Pax6 mutant, we examined expression patterns of Shh signalling molecules and states of cell death and cell proliferation. Shh was similarly expressed in the floor plate of the mutant hindbrain, while the expressions of Ptc1, Gli1 and Gli2 were altered only in the progenitor domains for the motoneurones. The position and number of TUNEL-positive cells were unchanged in the Pax6 mutant. Although the proportion of cells that were BrdU-positive slightly increased in the mutant, there was no relationship with specific progenitor domains. Taken together, we conclude that Pax6 regulates specification of the ventral neurone subtypes by establishing the correct progenitor domains.