Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation

AbstractBackground: The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation.Results Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh-transfected COS cells. Direct transfection of neural plate cells with an Shh-expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation.Conclusion The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors

Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus

BACKGROUND: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. RESULTS: In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (β-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. CONCLUSIONS: In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues

Crucial roles of Robo proteins in midline crossing of cerebellofugal axons and lack of their up-regulation after midline crossing

<p>Abstract</p> <p>Background</p> <p>Robo1, Robo2 and Rig-1 (Robo3), members of the Robo protein family, are candidate receptors for the chemorepellents Slit and are known to play a crucial role in commissural axon guidance in the spinal cord. However, their roles at other axial levels remain unknown. Here we examine expression of Robo proteins by cerebellofugal (CF) commissural axons in the rostral hindbrain and investigate their roles in CF axon pathfinding by analysing Robo knockout mice.</p> <p>Results</p> <p>We analysed the expression of Robo proteins by CF axons originating from deep cerebellar neurons in rodent embryos, focusing on developmental stages of their midline crossing and post-crossing navigation. At the stage of CF axon midline crossing, mRNAs of Robo1 and Robo2 are expressed in the nuclear transitory zone of the cerebellum, where the primordium of the deep cerebellar nuclei are located, supporting the notion that CF axons express Robo1 and Robo2. Indeed, immunohistochemical analysis of CF axons labelled by electroporation to deep cerebellar nuclei neurons indicates that Robo1 protein, and possibly also Robo2 protein, is expressed by CF axons crossing the midline. However, weak or no expression of these proteins is found on the longitudinal portion of CF axons. In <it>Robo1</it>/<it>2 </it>double knockout mice, many CF axons reach the midline but fail to exit it. We find that CF axons express Rig-1 (Robo3) before they reach the midline but not after the longitudinal turn. Consistent with this <it>in vivo </it>observation, axons elicited from a cerebellar explant in co-culture with a floor plate explant express Rig-1. In <it>Rig-1 </it>deficient mouse embryos, CF axons appear to project ipsilaterally without reaching the midline.</p> <p>Conclusion</p> <p>These results indicate that Robo1, Robo2 or both are required for midline exit of CF axons. In contrast, Rig-1 is required for their approach to the midline. However, post-crossing up-regulation of these proteins, which plays an important role in spinal commissural axon guidance, does not appear to be required for the longitudinal navigation of CF axons after midline crossing. Our results illustrate that although common mechanisms operate for midline crossing at different axial levels, significant variation exists in post-crossing navigation.</p

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters

乳がんの再発を起こす原因細胞を解明. 京都大学プレスリリース. 2023-11-16.The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain–containing ion transport regulator 3 (FXYD3), a component of the Na⁺/K⁺ pump. Accordingly, FXYD3⁺ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3⁺ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3⁺ CSCs were sensitive to senolytic Na⁺/K⁺ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3⁺ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na⁺/K⁺ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis

A Study of Clinical Factors for Predicting Suitable Treatment of Diabetes, and Factors Associated with Complications in Non-Insulin-Dependent Diabetes Mellitus (NIDDM)

糖尿病の治療方法を早期に決定し,持続性の高血糖状態を改善することは糖尿病性合併症の予防に重要と考えられるので,今回治療法の決定に関与すると考えられる因子について検討を行った。糖尿病の教育および治療目的で入院したインスリン非依存性糖尿病(non-insulln-dependent diabetes mellitus以下NIDDMと略す)患者436名を対象に,コントロール前の各臨床データより最終的治療法がどの程度予測可能か,また関与の大きい因子は何かを林の数量化理論第II類を用いた多変量解析により検討を加えた。目的変数は食事・運動療法(以下食事療法と略す),経口血糖降下剤療法(以下経口剤療法と略す),インスリン療法の各治療法とし,前もって単変量分析で関与が大きいと考えられた網膜症,腎症,神経症,肥満度,罹病期間,24時間蓄尿中のC-peptlde like immunoreactivity(以下U-CPRと略す)値,空腹時血糖値,75g経口ブドウ糖負荷試験(以下75gOGTTと略す)における120分,180分血糖値,およびヘモグロビンA1c、(以下HbA1cと略す)値の10因子を説明変数として多変量解析を行った。 その結果,1)治療法の選択に際し,まず考慮すべきは,肥満度,罹病期間,空腹時血糖値の3因子であり,続いて75gOGTTにおける120分血糖値およびU-CPR値であった。すなわち痩せて,罹病期間が長く,血糖コントロールが不良でインスリン分泌能が低い症例に対しては経口剤もしくはインスリン治療の必要性が大きいと考えられた。一方,網膜症や腎症などの合併症の因子の関連は低いものであった。2)以上の10因子を用いる事によって,食事療法群-経口剤療法群間,食事療法群-インスリン療法群間,経口剤療法群一-インスリン療法群間の判別的中率はそれぞれ87.9%,95%,60%であり,食事群と薬剤群との判別は良好であった。3)治療法を経口剤療法にするかインスリン療法にするのが適当かを決定するには今回用いた入院時の10因子のみでは困難であり,個々の症例により検討を行う必要がある。 次に,糖尿病合併症の進行を阻止するには合併症の進展に影響を及ぼす因子の解明が重要であると考えられる。そこで多変量解析の手法を同様に用いて糖尿病性網膜症,腎症,神経症,および心電図異常の各合併症に関与すると考えられる因子の解析を行った。目的変数は各合併症の有無やその程度とし,説明変数として網膜症,腎症,神経症,心電図異常,年齢,性,罹病期間,肥満度,治療法,最大血圧,最小血圧,空腹時血糖値,75gOGTTにおける120分血糖値,180分血糖値,尿酸値,血清総コレステロール値および中性脂肪値の各項目とした。その結果,1)網膜症,腎症,神経症の細小血管障害の共通関与因子は,罹病期間,肥満度,年齢であり,その発症・進展には共通のメカニズムが関与している可能性が示唆された、2)いずれの細小血管障害においても空腹時および糖負荷時の血糖値,性,心電図異常との関連は小さかった。3)入院時の血糖のコントロール状態と各合併症との関連は今回明かでなかったが,今後プロスペクティヴな検討が必要である。4)心電図異常を大血管障害の一指標とみなすと大血管障害と細小血管障害に関与する因子の共通性は少なく,それぞれの成因に異なった因子の関与が示唆された。5)大血管障害に影響を及ぼす困子はより多彩であり,今回用いた因子のみの分析では不充分と考えられた。This study was conducted to evaluate clinical factors for prediction of suitable treatment and to identify factors associated with complications using a statistical method of quantification theory II in 436 patients with NIDDM. The results were as follows; 1)The important factors for prediction of suitable treatment were fasting plasma glucose level, duration of diabetes and obesity index. Plasma glucose level at 120 min.on 75gOGTT and U-CPR level were also important. 2)Presence of complications such as retinopathy or nephropathy did not influence selection of treatment. 3)It was relatively easy to choose either diet therapy or drug therapies in NIDDM. However, it was difficult to select either oral-hypoglycemic agent or insulin therapy. 4) Retinopathy, nephropathy and neuropathy strongly influenced their development each other. Factors mutually affecting these diabetic microangiopathies were the duration of diabetes, obesity index and age. On the other hand, the concern of sex, fasting plasma glucose level and ECG abnormality were less related to the development of microangiopathy. 5)Factors affecting macroangiopathy, as indicated by ECG abnormality, differed from those of microangiopathy. Hence, further evaluation is needed for clarifying factors affecting the development of macroangiopathy.広島大学(Hiroshima University)博士(医学)Medicinedoctora

Avian adeno-associated virus vector efficiently transduces neurons in the embryonic and post-embryonic chicken brain.

The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits

A3V-mediated Tet-inducible system robustly transduced a sparse population of NM and NL neurons.

<p>(A) A3V-TRE-EGFP and A3V-RSV-rtTAV16 (total 0.5–1.5 µl, 5×10¹¹ GC/ml each) were injected at E3.5, and Dox was administered at E6.5. (B) Double immunofluorescence labeling for EGFP (green) and NeuN (red). No apparent EGFP signal was detected at E17 in the Dox (−) preparation (upper), while strong EGFP signal was observed both at E9 (middle) and E17 (bottom) in Dox (+) embryos. Scale bars indicate 100 µm. (C and D) Higher magnification views of EGFP-expressing NM neurons at E9 and E17, respectively. Scale bars indicate 20 µm. (E and F) NL neurons at E9 or E17, respectively. Scale bars indicate 20 µm.</p

A3V gene transduction in post-hatch chick brain.

<p>(A–C) EGFP expression 1 week after A3V injection (a total of 5×10⁷, 5×10⁸, and 5×10⁹ GC, respectively) was analyzed by immunofluorescence labeling of parasagittal sections. Arrowheads indicate injection sites in the striatum. Scale bars are 1 mm. (D) Gene transduction after LV or A3V injection was quantified by measurements of EGFP-expressing area in the parasagittal sections containing injection sites (n = 4). The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s004" target="_blank">Table S4</a>. (E) A3V-treated striatal cells were visualized by double immunofluorescence labeling for EGFP (green) and neuronal marker NeuN (red). Scale bar indicates 50 µm. (F) EGFP expression 24 hrs after A3V injection (a total of 5×10⁹ GC) into the intermediate medial mesopallium (IMM) was analyzed by immunofluorescence labeling of coronal sections. The arrowhead indicates the A3V injection site. Scale bar is 1 mm.</p

Comparison of transduction properties of A3V, AAV2 and LV.

<p>(A) Constructs of A3V-RSV-EGFP, AAV2-RSV-EGFP, and LV-RSV-EGFP. ITR, inverted terminal repeat; RSV, Rous sarcoma virus promotor; EGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; pA, SV40 polyadenylation signal; LTR, long terminal repeat; psi, packaging signal; RRE, Rev-responsive element; cPPT, central polypurine tract. (B) A representative example of EGFP-expressing cultured chicken neural cells after A3V treatment. Scale bar indicates 20 µm. (C–E) A3V-treated chicken neural cells, zebra finch neural cells, and 293T cells, respectively. Upper and lower panels represent the DAPI nuclear staining and EGFP fluorescent images of the same fields of view, respectively. All fluorescent images were taken with the same exposure time. (F–K) As a comparison, fluorescent images of corresponding cultured cells after AAV2 or LV treatment are shown. All images were taken with the same exposure condition as in (C–E). Scale bar indicates 100 µm. (L and M) Quantification of overall gene expression and transduction rate, respectively (n = 4). a.u., arbitrary units. *p<0.05; **p<0.005. The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s001" target="_blank">S2</a>.</p