非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究

神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发现，在爪蟾胚胎中Nkx6.3的过表达或抑制表达都导致神经嵴标记基因表达降低。进一步研究发现，32细胞期在不同部位注射Nkx6.3 mRNA可以异位诱导或抑制Slug的表达。动物帽实验显示，Nkx6.3单独过表达可以诱导神经嵴发生，而迄今为止转录因子中只有Snail1具有这一单独诱导能力。在爪蟾胚胎及动物帽中，过表达Nkx6.3都可以诱导Fgf8、Wnt8而抑制BMP4的转录，而且Nkx6.3对这些分泌因子的调控方式是不同的。4细胞期过表达Nkx6.3的胚胎，在促进Fgf8和Wnt8而抑制BMP4的同时，却抑制神经板边界特异基因Msx1、Pax3和神经嵴特异基因Slug的表达，说明Nkx6.3对神经嵴的诱导调控在神经板边界基因层次还存在抑制作用。32细胞过表达Nkx6.3会细胞自主性抑制以及细胞非自主诱导Msx1、Pax3、Slug的表达。Nkx6.3异位诱导Dlx5却抑制Dlx3的表达，说明Dlx5可能是Nkx6.3负调控的直接靶基因。由此，我们提出Nkx6.3的神经嵴诱导调控分为两个层次：分泌信号分子水平的正调控和神经板边界决定水平的负调控。在脊椎动物的神经发生过程中，神经管背腹不同层次形成不同的神经元。这些神经元细胞的命运由背腹起源的多种形态发生素决定。形态发生素通过浓度梯度确定了一组转录因子在神经管背腹不同层次的特异表达，这些基因的组合调控决定了神经前体细胞的命运。然而，这些转录因子是如何解读形态发生素梯度信号的还不是很清楚。第四章，我们通过对神经管腹侧特异表达的转录因子的调控区进行预测，确定了可能调控这些基因表达的保守区段。此外，我们改进了爪蟾转基因操作，并用这一技术确证了Nkx6.2的调控区域。Dbx1、Nkx2.2及Pax6的转录调控区已在小鼠或爪蟾中报道过。由此我们得到了两对在神经管背腹图式中相互作用的转录因子的调控区域：Nkx6.2和Dbx1、Nkx2.2和Pax6。通过对Nkx6.2和Dbx1的调控保守区的转录因子结合位点的预测，我们发现这四个基因以及Wnt信号之间存在大量的相互调控。然而在这两个基因的调控区，我们没有发现Gli的调控位点，暗示这两个基因可能不受Shh的直接调控。我们还克隆了Dbx家族的两个基因，并检测了它们的时空特异性表达，发现Dbx2是母源性表达的，而Dbx1是合子型基因。这两个基因的表达图式相似，都在神经板中线两侧成线状表达，尾芽期在神经管中部表达。过表达Dbx2抑制神经元的初级分化，说明它可能与Dbx1一样具有维持神经板细胞未分化状态的功能。Dbx2的过表达还抑制Nkx6.2及Dbx1的表达，说明它们可能一起参与了神经管腹侧图式的调控。

Investigation on the strain relaxation of InGaN layer and its effects on the InGaN structural and optical properties

The evolution of strain and structural properties of thick epitaxial InGaN layers grown on GaN with different thicknesses are investigated. It is found that, with increase in InGaN thickness, plastic relaxation via misfit dislocation generation becomes a more important strain relaxation mechanism. Accompanied with the relaxation of compressive strain, the In composition of InGaN layer increases and induces an apparent red-shift of the cathodoluminescence peak of the InGaN layer. On the other hand, the plastic relaxation process results in a high defect density, which degrades the structural and optical properties of InGaN layers. A transition layer region with both strain and In composition gradients is found to exist in the 450-nm-thick InGaN layer.

STRUCTURAL DEFECTS AND THEIR ELECTRICAL-ACTIVITY IN GERMANIUM IMPLANTED SILICON

The electrical and structural characteristics of secondary defects in regrown amorphous layers formed in n-type Si(100) with a resistivity of 2 OMEGA cm and 6 OMEGA cm using Ge+ ions, has been studied. The amorphous layers with a thickness of 460 nm are formed by implantation of 1 x 10(15) Ge+ cm-2 at an energy of 400 keV. Both conventional furnace and rapid thermal annealing were used to regrow the amorphous layer and the residual defects have been characterised in terms of their concentration depth distribution and activation energies using C-V and DLTS. Structural information has been obtained from RBS and XTEM. By choosing suitable anneal conditions it is possible to eliminate extended defects, apart from a low concentration of end of range dislocation loops. However, a substantial population of electrically active point defects remain after simple low thermal budget anneals. In a sample implanted with 1 x 10(15) Ge+ cm-2 at 400 keV a region of deep donors approximately 460 nm from the surface is always present When the samples are annealed at higher temperatures (> 850-degrees the total deep donor concentration is reduced by one order of magnitude. Other electrically active defects not observable in the low (750-degrees-C) temperature annealed layers become apparent during anneals at intermediate temperatures.

PRELIMINARY-RESULTS OF GAAS SINGLE-CRYSTAL GROWTH UNDER HIGH GRAVITY CONDITIONS

GaAs single crystals have been grown under high gravity conditions, up to 9g0, by a recrystallization method with decreasing temperature. The impurity striations in GaAs grown under high gravity become weak and indistinct with smaller striation spacings. The dislocation density of surcharge-grown GaAs increases with increase of centrifugal force. The cathodoluminescence results also show worse perfection in the GaAs grown at high gravity than at normal earth gravity.

DELINEATION OF DEFECTS IN SIMOX STRUCTURES USING A CHEMICAL ETCHING TECHNIQUE

Residual defects in the overlayer of fully annealed SIMOX material have been studied by means of a chemical etching technique. The etching procedure has been calibrated and an optimum recipe is reported. Observations using optical microscopy and transmission electron microscopy have been used to quantify the defect densities and good agreement between the two techniques has been established, confirming that the optimised chemical etching process can be used with confidence to determine the dislocation density for values < 10(7) cm-2.

Growth of high-quality relaxed In0.3Ga0.7As/GaAs superlattices

With a low strained InxGa1-xAs/GaAs(x similar to 0.01) superlattice (SL) buffer layer, the crystal quality of 50 period relaxed In0.3Ga0.7As/GaAs strained SLs has been greatly improved and over 13 satellite peaks are observed from X-ray double-crystal diffraction, compared with three peaks in the sample without the buffer layer. Cross-section transmission electron microscopy reveals that the dislocations due to superlattice strain relaxation are blocked by the SLs itself and are buried into the buffer layer. The role of the SL buffer layer lies in that the number of the dislocations is reduced in two ways: (1) the island formation is avoided and (2) the initial nucleation of the threading dislocations is retarded by the high-quality growth of the SL buffer layer. When the dislocation pinning becomes weak as a result of the reduced dislocation density, the SLs can effectively move the threading dislocations to the edge of the wafer.

Self-formed InGaAs/GaAs quantum dot superlattice and direct observation on strain distribution in the capped superlattice

We have demonstrated a 20 period dislocation-free InGaAs/GaAs quantum dot superlattice which is self-formed by the strain from the superlattice taken as a whole rather than by the strain from the strained single layer. The island formation does not take place while growing the corresponding strained single layer. From the variation of the average dot height in each layer, the strain distribution and relaxation process in the capped superlattice have been examined. It is found that the strain is not uniformly distributed and the greatest strains occur at two interfaces between the superlattice and the substrate and the cap layer in the capped superlattice. (C) 1997 American Institute of Physics.

Dislocations and precipitates in semi-insulating gallium arsenide revealed by ultrasonic Abrahams-Buiocchi etching

The dislocations and precipitates in SI-GaAs single crystals are revealed by ultrasonic-aided Abrahams-Buiocchi etching (USAB), and the etch pits are observed and measured by metalloscope and scanning electron microscope (SEM) equipped with an energy dispersive X-ray spectrometer (EDS), respectively. The size of etch pit revealed by USAB etching is about 1 order of magnitude smaller than that revealed by molten KOH. The amount of arsenic atoms in the dislocation-dense zone is about 1% larger than that in an adjacent dislocation-free zone measured by EDS attached to SEM, which indicates that the excess arsenic atoms adjacent to the dislocation-dense zone are attracted to the dislocations and precipitate there due to the deformation energy.

Characterization and improvement of GaAs layers grown on Si using an ultrathin a-Si film as a buffer layer

GaAs epilayers grown on Si by metalorganic chemical vapor deposition (MOCVD) using an ultrathin a-Si buffer layer were characterized by deep-level transient spectroscopy (DLTS). Six electron traps with activation energies of 0.79, 0.67, 0.61, 0.55, 0.53 and 0.32 eV below the conduction band were determined by fitting the experimental spectra. Two of the levels, C (0.61 eV) and F (0.32 eV), were first detected in GaAs epilayers on Si and identified as the metastable defects M3 and M4, respectively. In order to improve the quality of GaAs/Si epilayers, another GaAs layer was grown on the GaAs/Si epilayers grown using MOCVD. The deep levels in this regrown GaAs epilayer were also studied using DLTS. Only the EL2 level was found in the regrown GaAs epilayers. These results show that the quality of the GaAs epilayer was greatly improved by applying this growth process.

BREAKING UP OF MISFIT DISLOCATIONS IN GAASIN0.3GA0.7AS/GAAS HETEROSTRUCTURE

The effect of GaAs cap layer with different thicknesses in the GaAs/In0.3Ga0.7As/GaAs heterostructure on misfit dislocation is investigated with transmission electron microscopy, and it is found that lines of misfit dislocation break up and move out of the structure when the GaAs cap layer thickness exceeds a certain amount. The breaking up and moving out of misfit dislocations, initially confined in the (001) substrate/InGaAs epilayer interface, occur mainly along the [110] direction on the interface in the structure. (C) 1995 American Institute of Physics.

In situ annealing during the growth of relaxed SiGe

In this paper, a graded Si1-xGex buffer and thereafter the Si0.8Ge0.2 uniform layer were grown at a little lower temperature to keep the surface smooth, which will provide the gliding dislocations a wider channel and less low energy nucleation sites on the surface. Therefore, the dislocation density may be reduced. However, the motion of the existing threading dislocations cannot retain equilibrium at lower temperature, strain will accumulate and be in favor of the nucleation of dislocation. In situ annealing was used to reduce the residual strain in the sample during the low-temperature growth of SiGe. A fully relaxed Si0.8Ge0.2 layer was obtained with the surface dislocation density of 3x10(5)cm(-2).

A model of dislocations at the interface of the bonded wafers

Wafer bonding is regardless of lattice mismatch in the integration of dissimilar semiconductor materials. This technology differs from the heteroepitaxy mainly in the mechanism of generating dislocations at the interface. A model of dislocations at the bonded interface is proposed in this paper. Edge-like dislocations, which most efficiently relax the strain, are predominant at the bonded interface. But the thermal stress associated with large thermal expansion misfit may drive dislocations away from the bonded interface upon cooling.

Dislocations in InAs epilayers grown by MBE on GaAs substrates under various conditions

Using Transmission Electron Microscopy, we studied the misfit and threading dislocations in InAs epilayers. All the samples, with thickness around 0.5 mu m, were grown on GaAs(001) substrates by molecular beam epitaxy under As-rich or in-rich conditions. The As-rich growth undergoes 2D-3D mode transition process, which was inhibited under In-rich surface. High step formation energy under As-deficient reconstruction inhibits the formation of 3D islands and leads to 2D growth. The mechanism of misfit dislocations formation was different under different growth condition which caused the variation of threading dislocation density in the epilayers.

Size and shape evolution of self-assembled coherent InAs/GaAs quantum dots influenced by seed layer

The size and shape Evolution of self-assembled InAs quantum dots (QDs) influenced by 2.0-ML InAs seed layer has been systematically investigated for 2.0, 2.5, and 2.9-ML deposition on GaAs(1 0 0) substrate. Based on comparisons with the evolution of InAs islands on single layer samples at late growth stage, the bimodal size distribution of InAs islands at 2.5-ML InAs coverage and the formation of larger InAs quantum dots at 2.9-ML deposition have been observed on the second InAs layer. The further cross-sectional transmission electron microscopy measurement indicates the larger InAs QDs: at 2.9-ML deposition on the second layer are free of dislocation. In addition, the interpretations for the size and shape evolution of InAs/GaAs QDs on the second layer will be presented. (C) 2001 Elsevier Science B.V. All lights reserved.

New method for the growth of highly uniform quantum dots

A new method is realized for the growth of self-formed quantum dots. We identify that dislocation-free islands can be formed by the strain from the strained superlattice taken as a whole. Unlike the Stranski-Krastanow (S-K) growth mode, the islands do not form during the growth of the corresponding strained single layers. Highly uniform quantum dots can be self-formed via this mechanism. The low temperature spectra of self-formed InGaAs/GaAs quantum dot superlattices grown on a (001) GaAs substrate have a full width at half maximum of 26-34 meV, indicating a better uniformity of quantum dot size than those grown in the S-K mode. This method can provide great degrees of freedom in designing possible quantum dot devices. 1998 Published by Elsevier Science B.V. All rights reserved.