微量注射抗TGF_β-1抗体对爪蟾发育的影响

注射抗TGF_β-1抗体至2细胞胚胎的一个裂球中,产生了三个级别的卵黄团外露畸型胚胎,YE-1、YE-2和YE-3。它们的产生与抗体的注射量有关。它们的共同特征是注射后胚胎能正常发育至囊胚,卵黄团在注射一侧外露,外露部分无任何中胚层组织。高剂量注射时,主要产生YE-1级别的畸型胚胎,其中大部分在注射侧从头至尾不存在肌肉组织,中胚层发育受到了极大的影响。降低注射剂量后所产生的YE-2畸型胚胎,在注射侧有一定的肌肉组织,但明显小于未注射侧。YE-3是所受影响最小的畸型胚胎,YE-3 V 的注射侧产生了与未注射侧非常接近的肌肉组织。活体观察畸型胚胎的形成过程,发现不同级别的畸型胚胎与注射侧的原肠运动所受影响程度和范围有关。高剂量注射时,几乎整个注射侧的原肠运动受到了影响,由此产生了YE-1,而低剂量注射时,注射侧只有部分区域受到影响,因此产生了YE-3 v 和YE-3 d。无论是原肠运动受阻,还是中胚层发育受到影响,其原因可能是抗TGF_β-1抗体阻抑了中胚层诱导的过程,所以由此也进一步证实了TGF_β相关蛋白与中胚层诱导有关。     

多肽生长因子与中胚层诱导

近几年来,许多发育生物学家采用分子生物学手段对利用实验胚胎学方法所揭示的胚胎早期发育的重要现象进行了分析研究,其中关于中胚层诱导的分子及细胞生物学机制的研究获得了极有意义的进展。研究中胚层诱导虽然主要以两栖类动物胚胎为主,但实际上中胚层诱导现象存在于所有脊椎动物的早期胚胎中,     

微量注射抗TGFβ—1抗体对爪蟾发育的影响

In order to testify the function of the TGF beta related protein, we microinjected the antibodies against the TGF beta-1 to one blastomere of two-cell stage embryos. Dosages of antibodies injected were 24-36 ng, 12 -18 ng, 6-9 ng and 1.5-2.25 ng. Malformed embryos with exposed yolk mass were produced. They were designated as YE-1, YE-2 and YE-3 according to the degree of malformation which was found to be related to the dosage of antibodies injected to the blastomere. These malformed embryos could normally develop to blastula, but the yolk mass was exposed on the injection half during gastrulation. In the group with the highest dosage, the most seriously affected embryos belong to the YE-1. For most of them, no muscle tissue could be found from head to tail in the injection half, the development of mesoderm seemed to be thoroughly inhibited. YE-2 was the group with decreased dosage, small block of muscle tissue was usually observed in the injection half. YE-3 was the least affected group with the injection half containing blocks of muscle tissue similar to that of the non-injection half. The different groups of malformations seemed to be related to the degree and range of interference on gastrulation. In the group with highest dosage, the gastrulation of the entire injection half seemed to be prohibited, and YE-1 was produced. In the groups with lower dosages, only part of injection half was interfered, and YE-3 v and YE-3 d were resulted. From the results mentioned above, we concluded that TGF beta-related protein are not only present in the early embryos of Xenopus laevis, but also may be concerned with mesoderm induction.     

TGF_β相关mRNAs在爪蟾早期胚胎中的表达

用TGF_β-1 cDNA片段作为探针,在爪蟾早期胚胎中检测到了能同此探针杂交的转录产物,分子量分别为4.2 kb,3.2 kb和2.3 kb,其中4.2 kb和3.2 kb的mRNA 在囊胚期(分期7—8)中的含量最高,而在卵裂期胚胎,原肠胚以及神经胚中含量极微。但2.3 kb的mRNA以卵裂期胚胎到神经胚中含最比较稳定。我们称这些能同TGF_β-1cDNA 探针杂之的mRNAs为TGF_β相关的mRNAs (TGFB_β-relatedmRNAs),它们在囊胚中主要分布在植物性半球,但背方和腹方的含量无明显差别。根据TGF_β相关mRNAs 在囊胚国的含量最高和主要分布在植物性半球,我们推测它们可能与中胚层诱导有关。     

TGFβ相关蛋白在爪蟾早期胚胎中的免疫组织化学的研究

By the method of immunocytochemistry, using the polyclonal antibodies raised against the 1-29 N-terminal residues of TGF beta-1, we found that the protein could bind to the antibodies was present in the early embryos of Xenopus. The protein was named TGF beta-related protein. It was distributed mainly in the endoderm from blastula (stg. 7) to late neurula. In the blastula (stg. 8), the protein was localized in the vegetal hemisphere near the floor of the blastocoel [Plate I, Fig. 1]. In the early gastrula (stg. 10.5) [Plate I, Fig. 2], it was localized in the central part of the vegetal hemisphere. In late gastrula (stg. 12), it was mainly distributed around the gastrocoel [Plate I, Fig. 3], but the fluorescence in endoderm cells (ventral part beneath the gastrocoel) was stronger than in the mesoderm cells (dorsal part of the gastrocoel). In the early neurula (stg. 14), the whole endoderm displayed strong fluorescence and the part of dorsal mesoderm (presumptive somite & notochord) close to endoderm was also found to be positively stained [Plate I, Fig. 4,5], but the part close to neural plate was negative. In The late neurula (stg. 20) [Plate I, Fig. 6], it was found in the central area of yolk mass (endoderm cells). No positive stain was detected in the unfertilized egg, embryos earlier than stage and later than stage 20/21.2+ protein in early development.     

TGF_β相关蛋白在爪蟾早期胚胎中的免疫组织化学的研究

在爪蟾早期胚胎中,存在能与抗TGF_β-1抗体结合的蛋白,我们称之为TGF_β-相关蛋白。用免疫细胞化学方法,检测到在囊胚(分期8)中,TGF_β相关蛋白主要分布在植物半球靠近囊胚腔底部的一些细胞内;在早期原肠胚(分期10.5)中仍只分布在植物半球卵黄团中,但距囊胚腔较远,与背唇无明显的关系;在晚期原肠胚(分期12)中,它主要分布在原肠腔四周,原肠腔腹方的内胚层细胞中含量高于原肠腔背方的中胚层细胞;在早期神经胚中(分期14),整个内胚层细胞都分布有TGF_β相关蛋白,而且它还存在于背部中胚层(预定体节和脊索)中,但分布不均匀,靠近原始肠腔的部分含有TGF_β相关蛋白,而靠近神经板的部分则不具有它;在晚期神经胚(分期20)中,它仅分布在卵黄团中央区域的内胚层细胞内。点免疫测定的结果印证了以上的观察。此外,还断定从卵裂期到分期27的胚胎,存在着两个TGF_β相关蛋白的高合成区域。第一个区间为分期7至分期8;第二个区间为分期12至分期14。在分期14胚胎中,它的含量最高。根据TGF_β相关蛋白在胚胎中分布的时空特性,讨论了它可能的功能。     

中胚层诱导中反应系统的分子生物学研究

中胚层诱导机制的研究大体上应该包括两方面的内容:“诱导”本身的机制,即植物半球细胞是通过怎样的方式诱导动物半球细胞的;目前在这个领域中,主要是对诱导因子的研究。另外是对反应系统的研究,即动物半球细胞受到诱导后,所发生的一系列的变化,还包括对反应能力(competence)的研究等等。第一个方面的内容已在另文中作了较为详细的论述,本文只着重讨论近几年来对反应系统的研究成果。