Bikas Dimitrios V, Vardabasso Sara, Quickstad Gabrielle, Shpargel Karl B
bioRxiv. 2025 May 15:2025.05.15.654100. doi: 10.1101/2025.05.15.654100.
SOX9 and RUNX2 are lineage defining transcription factors that drive differentiation of chondrocyte and osteoblast lineages respectively from osteochondral progenitors. In limb development, these progenitors are specified first by SOX9 expression required for mesenchymal stem cell (MSC) condensation prior to RUNX2 activation and osteochondral differentiation to chondrocyte and osteoblast lineages. Unlike limb development, the anterior craniofacial skeleton arises from cranial neural crest (cNCC) stem cells. To examine the temporal activation of SOX9 and RUNX2 within cNCCs, we utilized a combination of immunofluorescence to detect endogenous proteins and genetic reporters to label SOX9 and RUNX2 expressing cells. We find that RUNX2 is expressed broadly throughout cNCC stem cells of the first branchial arch that will give rise to developing mandibular tissue at a timepoint prior to osteochondral lineage determination. Substantial SOX9 expression is activated subsequently within differentiating chondrocytes. These findings were validated by fluorescent reporters inserted in the 3' untranslated regions (3'UTRs) of and . Although the GFP based reporter did not delete any 3'UTR sequences, homozygous pups develop postnatal deficiencies in intramembranous and endochondral ossification that correlate with enhanced expression of RUNX2 protein in osteoblasts and hypertrophic chondrocytes. phenotypes model the human disorder, Metaphyseal Dysplasia with Maxillary Hypoplasia and Brachydactyly (MDMHB), resulting from RUNX2 enhanced activity due to intragenic duplications. Altogether, this reporter model provides a valuable tool for studying RUNX2 function in early cNCC-derived stem cell lineages and highlights the high sensitivity of ossification pathways to RUNX2 dosage.
We have developed a novel mouse model for a human disorder resulting from excessive RUNX2, a transcription factor required for bone formation. We find that RUNX2 turns on early within facial stem cells in a pattern unique from limb development. Excessive RUNX2 is particularly detrimental to bone growth in juvenile development after birth.
SOX9和RUNX2是谱系定义转录因子，分别驱动软骨细胞和成骨细胞谱系从骨软骨祖细胞分化而来。在肢体发育中，这些祖细胞首先通过间充质干细胞（MSC）凝聚所需的SOX9表达来确定，然后才激活RUNX2并向软骨细胞和成骨细胞谱系进行骨软骨分化。与肢体发育不同，前颅面部骨骼起源于颅神经嵴（cNCC）干细胞。为了研究cNCCs中SOX9和RUNX2的时间激活情况，我们结合使用免疫荧光检测内源性蛋白质和基因报告基因来标记表达SOX9和RUNX2的细胞。我们发现，RUNX2在第一鳃弓的整个cNCC干细胞中广泛表达，这些干细胞将在骨软骨谱系确定之前的一个时间点产生发育中的下颌组织。随后，在分化的软骨细胞中大量激活SOX9表达。这些发现通过插入 和 3'非翻译区（3'UTR）的荧光报告基因得到验证。尽管基于GFP的 报告基因没有删除任何3'UTR序列，但纯合 幼崽在出生后膜内和软骨内骨化方面出现缺陷，这与成骨细胞和肥大软骨细胞中RUNX2蛋白表达增强相关。 表型模拟了人类疾病——伴有上颌骨发育不全和短指（趾）的干骺端发育异常（MDMHB），该疾病是由基因内重复导致RUNX2活性增强引起的。总之，这个报告基因模型为研究RUNX2在早期cNCC衍生干细胞谱系中的功能提供了一个有价值的工具，并突出了骨化途径对RUNX2剂量的高敏感性。
我们开发了一种新型小鼠模型，用于研究因RUNX2过多导致的人类疾病，RUNX2是骨形成所需的转录因子。我们发现RUNX2在面部干细胞中早期开启，其模式与肢体发育不同。出生后过量的RUNX2对幼年发育中的骨骼生长特别有害。
