Chiyoko Kobayashi

The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development

Mutations in SALL4, the human homolog of the Drosophila homeotic gene spalt (sal), cause the autosomal dominant disorder known as Okihiro syndrome. In this study, we show that a targeted null mutation in the mouse Sall4 gene leads to lethality during peri-implantation. Growth of the inner cell mass from the knockout blastocysts was reduced, and Sall4-null embryonic stem (ES) cells proliferated poorly with no aberrant differentiation. Furthermore, we demonstrated that anorectal and heart anomalies in Okihiro syndrome are caused by Sall4 haploinsufficiency and that Sall4/Sall1 heterozygotes exhibited an increased incidence of anorectal and heart anomalies, exencephaly and kidney agenesis. Sall4 and Sall1 formed heterodimers, and a truncated Sall1 caused mislocalization of Sall4 in the heterochromatin; thus, some symptoms of Townes-Brocks syndrome caused by SALL1 truncations could result from SALL4 inhibition.

STRUCTURE OF THE PLANARIAN CENTRAL NERVOUS SYSTEM (CNS) REVEALED BY NEURONAL CELL MARKERS

Abstract Planarians are considered to be among the most primitive animals which developed the central nervous system (CNS). To understand the origin and evolution of the CNS, we have isolated a neural marker gene from a planarian, Dugesia japonica, and analyzed the structure of the planarian CNS by in situ hybridization. The planarian CNS is located on the ventral side of the body, and composed of a mass of cephalic ganglions in the head region and a pair of ventral nerve cords (VNC). Cephalic ganglions cluster independently from VNC, are more dorsal than VNC, and form an inverted U-shaped brain-like structure with nine branches on each outer side. Two eyes are located on the dorsal side of the 3rd branch and visual axons form optic chiasma on the dorsal-inside region of the inverted U-shaped brain. The 6th–9th branches cluster more closely and form auricles on the surface which may function as the sensory organ of taste. We found that the gross structure of the planarian CNS along the anterior-posterior (A–P) axis is strikingly similar to the distribution pattern of the “primary” neurons of vertebrate embryos which differentiate at the neural plate stage to provide a fundamental nervous system, although the vertebrate CNS is located on the dorsal side. These data suggest that the basic plan for the CNS development along the A–P axis might have been acquired at an early stage of evolution before conversion of the location of the CNS from the ventral to the dorsal side.

FGFR-related gene nou-darake restricts brain tissues to the head region of planarians

The study of planarian regeneration may help us to understand how we can rebuild organs and tissues after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well-organized central nervous system. In recent years, methodologies such as whole-mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. Here we report the identification and characterization of nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation was suppressed by inhibition of two planarian FGFR homologues (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signalling in Xenopus embryos. The data suggest that ndk may modulate FGF signalling in stem cells to restrict brain tissues to the head region of planarians.

To be or not to be a flatworm : the acoel controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.

IDENTIFICATION OF TWO DISTINCT MUSCLES IN THE PLANARIAN DUGESIA JAPONICA BY THEIR EXPRESSION OF MYOSIN HEAVY CHAIN GENES

Abstract Ultrastructural and physiological studies have shown that planarian muscles have some characteristics of smooth and some characteristics of striated muscles. To characterize planarian muscles, we isolated two myosin heavy chain genes (DjMHC-A and DjMHC-B) from a planarian, Dugesia japonica, by immunological screening, and analyzed their structures and spatial expression patterns. Structural analysis indicated that both MHC genes are striated-muscle-type myosin genes, although planarian muscles do not have any striation. In situ RNA hybridization showed that expression of the two myosin genes is spatially strictly segregated. DjMHC-A was expressed in pharynx muscles, pharynx cavity muscles, muscles surrounding the intestinal ducts, a subpopulation of body-wall muscles and several muscle cells in the mesenchymal region around the base of the pharynx. DjMHC-B was expressed in body-wall muscles (including circular, diagonal and longitudinal muscles), vertical muscles and horizontally oriented muscles. Double staining with DjMHC-A and -B probes clearly demonstrated that expression of the DjMHC-A and -B genes do not occur in the same cell. During regeneration, the number of cells positive for expression of each gene increased in the blastema region, suggesting that both types of muscle may be involved in blastema formation. DjMHC-B-positive cells disappeared from the body-wall muscle layer in the pharynx-cavity-forming region, whereas DjMHC-A-positive cells were markedly accumulated there, suggesting that the two types of muscle in the body wall layer may have distinct functions. These results indicate that planarians have at least two types of muscle that express striated-muscle-type MHC genes, but do not form striation.

Kif26b, a kinesin family gene, regulates adhesion of the embryonic kidney mesenchyme.

The kidney develops through reciprocal interactions between two precursor tissues: the metanephric mesenchyme and the ureteric bud. We previously demonstrated that the zinc finger protein Sall1 is essential for ureteric bud attraction toward the mesenchyme. Here, we show that Kif26b, a kinesin family gene, is a downstream target of Sall1 and that disruption of this gene causes kidney agenesis because of impaired ureteric bud attraction. In the Kif26b-null metanephros, compact adhesion between mesenchymal cells adjacent to the ureteric buds and the polarized distribution of integrin α8 were impaired, resulting in failed maintenance of Gdnf, a critical ureteric bud attractant. Overexpression of Kif26b in vitro caused increased cell adhesion through interactions with nonmuscle myosin. Thus, Kif26b is essential for kidney development because it regulates the adhesion of mesenchymal cells in contact with ureteric buds.

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the ‘neoblast’. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin‐like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1‐expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X‐ray‐irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1‐expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.

Notch2 Activation in the Embryonic Kidney Depletes Nephron Progenitors

Successive activation of Wnt4 and Notch2 generates nephrons from the metanephric mesenchyme. Mesenchymal-to-epithelial transition requires Wnt4, and normal development of the proximal nephron (epithelia of glomeruli and proximal tubules) requires Notch2. It is unknown, however, whether Notch2 dictates the fate of the proximal nephron directly. Here, we generated a mutant strain of mice with activated Notch2 in Six2-containing nephron progenitor cells of the metanephric mesenchyme. Notch2 activation did not skew the cell fate toward the proximal nephron but resulted in severe kidney dysgenesis and depletion of Six2-positive progenitors. We observed ectopic expression of Wnt4 and premature tubule formation, similar to the phenotype of Six2-deficient mice. Activation of Notch2 in the progenitor cells suppressed Pax2, an upstream regulator of Six2, possibly through Hesr genes. Taken together, these data suggest that a positive feedback loop exists between Notch2 and Wnt4, and that Notch2 stabilizes, rather than dictates, nephron fate by shutting down the maintenance of undifferentiated progenitor cells, thereby depleting this population.

Ectopic pharynxes arise by regional reorganization after anterior/posterior chimera in planarians.

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.

Trb2, a mouse homolog of tribbles, is dispensable for kidney and mouse development

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.