A refined in ovo window model for advanced stage chick embryos

The chick embryo window model uniquely offers direct access to the developing embryo via a window cut into the eggshell. This model has enabled impactful discoveries in developmental biology, angiogenesis, tumor growth, and more. Conventionally, a window is made along the long axis of the egg by incubation day 3 (D3) in a process referred to here as "side-windowing", which enables access to the embryo for chemical or physical manipulations. Recently, the chick embryo has become more frequently utilized for musculoskeletal development studies involving embryo manipulations at advanced developmental stages. Unfortunately, embryos in side-windowed eggs are challenged by rapidly decreasing viability and increasingly altered development over time, potentially due to the need to withdraw albumen and cease rocking to prevent spilling albumen through the window. Notably, side-windowing cannot be performed later than D3 to avoid damaging the chorioallantoic membrane, which adheres to the eggshell thereafter. An "aircell-window" model, in which a window is cut over the aircell at more advanced developmental stages, and without removing albumen, could potentially overcome these drawbacks. We demonstrated the feasibility of aircell-windowing at later developmental stages and determined that aircell-windowing at a more advanced developmental stage led to similar viability and musculoskeletal development with relatively few developmental differences from normal controls. Thus, the late-stage aircell-window model is an advantageous platform for studies with chick embryos requiring manipulations at more advanced stages.