Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

肌肉，肌腱及其附着部位的协调发展可确保出现功能性肌肉骨骼单位，以适应不同物种之间不同的解剖学要求。人们对在胚胎发生过程中如何对这些不同的组织进行构图和功能组装知之甚少。在这里，我们研究了眼外肌（EOMs）的形态发生，眼外肌是一种进化的保守性颅骨肌群，对于眼球的协调运动和视力至关重要。通过沿袭分析，我们重新定义了与EOM相互作用的眼周结缔组织的细胞起源，这并非像先前所认为的那样仅源于神经rest间充质。使用3D成像方法，我们为EOM功能单元建立了完整的蓝图。通过这样做，我们确定了一个发育时间窗口，其中单个EOM从独特的肌肉损伤中出现并在巩膜中建立插入，这使这些肌肉与经典的肌肉到骨骼类型的插入不同。此外，我们证明眼球是可扩散的全反式视黄酸（ATRA）的来源，它允许EOM以时间和剂量依赖性的方式靶向它们。使用转基因的小鼠和抑制剂治疗，我们发现类维生素A浓度的内源性局部变化有助于在肌肉图案形成之前建立肌腱缩合和附着位点。总的来说，