Protein kinase A regulates GDNF/RET-dependent but not GDNF/Ret-independent ureteric bud outgrowth from the Wolffian duct

Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression.     

The role of GDNF in patterning the excretory system

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.     

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.     

Florigen is involved in axillary bud development at multiple stages in Arabidopsis

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.     

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.     

Effects of digit tissue on cell death and ectopic cartilage formation in the interdigital zone in chick leg buds

In developing chick leg buds, large-scale cell death occurs in the interdigital zone, which is responsible for the separation of digits from each other. Ectopic cartilage formation is known to occur upon removal of the chondrogenic digit tissue of the leg bud. To examine the mechanisms of ectopic cartilage formation in the interdigital cell death region, we performed the following operations on stage 28–29 leg buds: (i) removal of the digit-forming area; (ii) incision between the interdigital zone and digit region; (iii) insertion of an aluminum barrier into the interdigital zone; and (iv) insertion of a permeable Nuclepore filter into the interdigital zone. In all cases, the inhibition of cell death and/or the formation of ectopic cartilage in the interdigital zone were observed, although the frequency of the inhibition of cell death and the formation of ectopic cartilage varied, depending upon the position where the operations were performed. These results suggest that cell death and cell differentiation in the interdigital zone may be controlled by some factor(s) from digit cartilage.     

Role of Wnt5a-Ror2 Signaling in Morphogenesis of the Metanephric Mesenchyme during Ureteric Budding

Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

香港四照花花芽分化的形态学观察

采用石蜡切片技术和形态观察对香港四照花(Dendrobenthamia hongkongensis(Hemsl.)Hutch.)花芽分化过程中花芽的形态变化进行观测,研究花芽外部形态与花芽分化之间的关系。结果显示,香港四照花的花芽分化开始于7月上旬,到9月底完成,形态分化过程可分为8个时期:未分化期、花序原基分化期、小花原基分化期、花萼原基分化期、花瓣原基分化期、雄蕊原基分化期、雌蕊原基分化期、雌蕊雄蕊形成期。与之对应的外部形态变化为:混合芽闭合,混合芽基部膨大,新叶展开露出圆形花序,花柄初现,花序膨大,花序表面小花突起,花柄伸长至4~6 mm,花序表面小花轮廓明显。香港四照花花芽外部形态能直观地反映出内部结构变化,可根据花芽外部形态特征推测花芽分化状况。研究结果可为香港四照花花期调控和栽培管理提供科学依据。     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

胚胎肾分支形态发生调控因素

胚胎肾发育最初阶段是中肾导管尾端在胶质细胞源性神经营养因子诱导下向背侧长出输尿管芽,而后成纤维细胞生长因子、肝细胞生长因子、骨形成蛋白、基质金属蛋白酶、整合素和粘附分子相继表达,作用于输尿管芽和间充质细胞,诱导分支形态发生,包括输尿管芽向间充质侵入、延伸以及间充质细胞向上皮转化。上述这些分子在功能上存在部分重叠与拮抗,维持细胞增殖和分化的平衡,从而保证输尿管芽形成正常的分支结构。本文对肾脏发育时期分支形态发生的调控因素进行综述。     

Another evidence for inhibitory effect of auxin in adventitious bud formation of decapitated flax (Linum usitatissimum L.) seedlings

Adventitious buds were formed on the hypocotyls of decapitated flax seedlings. Scanning electron and light microscopic examinations of hypocotyls showed that epidermal cells divided to produce meristematic spots from which several leaf primordia were formed. Between leaf primordia and the original vascular tissues of hypocotyls, new xylem cells were formed which connected them. About 10, 30 and 60% of adventitious buds were formed on upper, middle and basal parts of hypocotyls of decapitated seedlings, respectively. Removal of apical meristem together with longer hypocotyl zero to four cm long below the apical meristem) induced higher percentage of adventitious bud formation in the remaining hypocotyl. When the entire hypocotyl was cut into 16 segments (0.25 cm each) and these segments were cultured on MS medium containing 3% sucrose and 0.8% agar, adventitious buds were mainly formed in the lowest five segments. These results suggested that there was a gradient of inhibitory factor(s) from apical to basal part of hypocotyl with respect to adventitious bud formation. Auxin transport inhibitors, morphactin and TIBA induced adventitious bud formation on intact seedlings by suppressing the basipetal movement of auxin.     

VARIATION IN HYDRA'S TENTACLE NUMBERS AS A FUNCTION OF TEMPERATURE

Chlorohydra uiridissima whose tentacle number is altered at different temperatures, was studied to see how other developmental variables changed as a function of temperature. The results suggest that temperature is instrumental in establishing the size of bud and tentacle primordia, but the number of primordia present may play a limiting role.

Animals were cultured at 18, 23 and 28°C and shifted between the extreme temperatures. Large animals with 8 tentacles, small animals with 5 tentacles, and intermediate animals with 6 and 7 tentacles served as parents. Buds and parents were monitored daily and scored for numbers of buds and tentacles.

Temperature, not parental size, determined the size of the buds. At the lower temperature buds were produced more slowly and initiated less frequently, but occurred in greater numbers per parent and had more tentacles than at the higher temperatures. The duration of bud development also increased at lower temperature, but at the lowest temperature the duration of bud development was not correlated with tentacle numbers on buds.

Changes in the frequency of bud initiation and the duration of bud development induced by changing temperature did not parallel changes in the number of tentacles produced on buds. Animals shifted from 18°C to 28°C underwent rapid increases in the rate of bud initiation and rapid shortening in the duration of bud development, while animals shifted from 28°C to 18°C underwent equally rapid changes in the opposite directions. The number of tentacles produced on buds, however, changed slowly to that characteristic of buds acclimated to the new temperatures. The frequency of bud initiation and the duration of bud development, therefore, do not determine tentacle number.

The number of tentacles already present seems to limit possibilities for adding new tentacles. Parents with five tentacles were especially likely to undergo upward changes in their tentacle number while parents with eight tentacles were resistant to such changes.     

麝香石竹花芽离体发育的阶段控制

在离体条件下,利用不同的培养基对麝香石竹顶芽外植体的花芽发育进行了阶段控制的研究.结果表明,麝香石竹的顶芽外植体在MS+KT1.0mg/L+IAA1.0mg/L+蔗糖3.0%+琼脂0.8%的Ⅰ级培养基上能被诱导花芽发育的启动;然后,将已诱导花芽发育启动的顶芽外植体,转接到MS+KT1.0mg/L+IAA0.5mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅱ级培养基上能进行花芽的进一步发育形成花蕾,且能从一个花蕾继续分化发育重新产生2—3个花蕾;把花蕾再转接到改良的MS+BA2.0mg/L+NAA0.2mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅲ级培养基上,培养一周后花蕾的花瓣张开,花朵全部开放.不同麝香石竹品种,诱导花芽发育启动的效果不同,Scania品种诱导效果最好.花芽发育初期可溶性蛋白含量较高,但随着花芽发育的进程而迅速下降,不同花芽发育时期的过氧化物酶活性均强于营养器官.本文为花芽分化发育机理的研究创造了条件,也为鲜花生产探索了新路子.     

Evidence that cytokinin controls bud size and branch form in Norway spruce

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current-year whorl shoots of 15- to 20-year-old trees of Norway spruce [ Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.
We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.     

黄花杓兰的花芽发育

对黄花杓兰（Cypripedium flavum P.F.Hunt et Summerh.）成年植株做了一个生长季的研究,提出了一年芽、二年芽和多年休眠芽的概念。指出由芽形成到植株开花需两年时间,其具体发育路线是：第一年6－7月份,根状茎顶端二年芽基部外侧有两个新的小芽产生,即“一年芽”,至9－10月份发育出7－9片幼叶,然后随气温下降停止生长;第2年4月份复苏,即为“二年芽”,二年芽在本生长季内发育成混合芽,但一般情况下只有一个充分发育,另一个未能充分发育并且一般将来也不再有发育的机会,被称为“多年休眠芽”;第3年5月份充分发育的二年芽长出地面,形成植株,迅速开花、结果,至9月底植株枯萎。本文还讨论了黄花杓兰发育过程与环境的关系。     

Evidence that cytokinin controls bud size and branch form in Norway spruce

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current-year whorl shoots of 15- to 20-year-old trees of Norway spruce [ Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.
We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.     

Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice

Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCγ, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT.     

低营养条件下Paclobutrazol(PP333)对黄瓜去顶幼苗直接形成花芽的影响(简报)

植物开花机理是生物学中的一个基本问题,多年来人们进行过许多的研究,积累了大量的事实,然而对开花的机理仍然还不甚清楚。因而在利用原有实验系统的同时,有必要寻找更多简单,又便于分析的实验系统。Jullien等报告离体培养的大豆子叶节能直接产生花芽。我们在建立离体培养黄瓜子叶直接单独形成雄花或雌花的实验系统的过程中,发现黄瓜幼苗去除顶芽后在子叶节处也能直接形成花芽。这一现象有可能用于深入研究各营养器官和花启动间关系等问题,定将