Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA; Developmental Biology Center, University of Minnesota, Minneapolis, MN, USA.
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA.
Dev Biol. 2023 Sep;501:28-38. doi: 10.1016/j.ydbio.2023.06.004. Epub 2023 Jun 8.
Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.
最近的研究表明，细胞代谢，尤其是糖酵解和糖酵解分支途径的调节，在脊椎动物胚胎发育过程中非常重要。例如，糖酵解产生细胞能量 ATP。葡萄糖碳也被导向戊糖磷酸途径，该途径对于快速生长的胚胎中的合成代谢过程是必需的。然而，我们对糖酵解代谢的确切状态以及调节糖酵解代谢的基因的理解仍然不完整。Sall4 是一种锌指转录因子，在发育中的小鼠胚胎中未分化的细胞中高度表达，例如胚泡和着床后的外胚层。TCre;Sall4 条件性基因敲除小鼠胚胎在后体部分表现出各种缺陷，包括后肢。使用转录组学方法，我们发现许多编码糖酵解酶的基因在后轴，包括后肢形成区域，在 Sall4 条件性基因敲除小鼠胚胎中上调。原位杂交和 qRT-PCR 也证实了几个糖酵解基因在后肢芽中的表达上调。其中一些基因的启动子、基因体或远端区域被 SALL4 结合，表明 Sall4 直接调节后肢芽中几个糖酵解酶基因的表达。为了进一步深入了解与转录水平观察到的变化相关的代谢状态，我们通过高分辨率质谱对野生型和 Sall4 条件性基因敲除胚胎的肢芽中的代谢物水平进行了全面分析。我们发现糖酵解中间产物的水平较低，但 Sall4 条件性基因敲除后肢芽中的糖酵解终产物丙酮酸和乳酸没有差异。糖酵解基因的高表达会导致糖酵解流加速，从而导致中间产物水平降低。这种情况可能阻止了中间产物重新定向到其他途径，例如戊糖磷酸途径。事实上，糖酵解代谢物水平的变化与 ATP 水平降低和戊糖磷酸途径代谢物水平降低有关。为了进一步测试糖酵解是否调节 Sall4 下游的肢体模式形成，我们条件性地使 Hk2 失活，Hk2 编码糖酵解的限速酶基因，受 Sall4 调节。TCre;Hk2 条件性基因敲除后肢表现出股骨短，后肢胫骨和前趾缺失，这与 TCre;Sall4 条件性基因敲除后肢的缺陷相似。Sall4 突变体和 Hk2 突变体的骨骼缺陷相似表明，糖酵解的调节在肢体模式形成中起作用。这些数据表明，Sall4 限制了肢芽中的糖酵解，并有助于肢芽发育过程中葡萄糖碳流的模式形成和调节。
