Supplementary Material for: Spatiotemporal characterisation of key cortical developmental markers in the developing ferret brain

Introduction
The ferret is an important model for studying corticogenesis and cortical gyrification due to its small size, condensed cortical development timeline, and postnatal onset of gyrification. Its cortical progenitor and neuronal diversity closely resemble those of humans. However, detailed histological data across the rostrocaudal axis at critical embryonic and postnatal stages remain limited, particularly for recently identified progenitor subpopulations. This study aimed to comprehensively characterise the spatiotemporal expression of key progenitor and neuronal markers throughout the rostrocaudal axis of the developing ferret cortex at critical embryonic and postnatal ages. In doing so, the study sought to establish a foundational, descriptive atlas of neurodevelopmental marker expression across key time points and cortical regions and layers.
Methods
Immunofluorescent labelling of key neural progenitor and neuronal markers was performed on coronal ferret brain sections at embryonic (E34, E38) and postnatal (P2, P5, P15, P25) ages. Markers included PAX6, SOX2, TBR2, HOPX, CPLX3, CTIP2, SATB2, TUJ1, and DCX. Semi-quantitative analyses described the spatiotemporal distribution of each marker within defined cortical compartments along the rostrocaudal axis.
Results
Early radial glial markers PAX6 and SOX2 were abundant in the ventricular zone at embryonic stages, progressively declining postnatally as the subventricular zone (SVZ) expanded. Intermediate progenitor cells labeled by TBR2 showed high abundance in the SVZ prenatally, with a marked decrease after birth. HOPX identified outer radial glia populations exhibiting distinct temporal and spatial distributions, with increasing presence in the subplate (SP) and cortical plate during postnatal stages. CPLX3 expression emerged postnatally, delineating mature SP neurons with regionally patterned maturation. Deep- and superficial-layer neuronal markers CTIP2 and SATB2 displayed orderly laminar emergence, indicating progressive cortical layer formation. General neuronal markers TUJ1 and DCX highlighted the maturation and migration of postmitotic neurons, with spatiotemporal gradients reflecting cortical differentiation across regions.
Discussion
This detailed profiling fills critical gaps in the ferret histological record and serves as a valuable resource for investigations into mammalian corticogenesis using the ferret model. Through the integration of semi-quantitative assessments and qualitative analysis, this dataset contributes to the ongoing development of a detailed atlas of ferret brain development. These findings are expected to enhance the utility of the ferret model in neurodevelopmental research, particularly in translational contexts involving human cortical malformations.

引言
雪貂是研究皮层发生（corticogenesis）与皮层回旋（cortical gyrification）的重要动物模型，其优势在于体型小巧、皮层发育周期紧凑，且皮层回旋过程始于出生后阶段。该物种的皮层祖细胞与神经元多样性与人类高度相似，但目前胚胎及出生后关键阶段中，沿吻尾轴（rostrocaudal axis）的详细组织学数据仍较为匮乏，针对新近鉴定的祖细胞亚群更是如此。本研究旨在全面表征发育中的雪貂皮层在胚胎及出生后关键时间点内，沿吻尾轴的关键祖细胞与神经元标志物的时空表达模式，借此建立覆盖关键时间节点、皮层区域与皮层层的神经发育标志物表达的基础性描述图谱。

研究方法
对胚胎期（E34、E38）及出生后（P2、P5、P15、P25）的雪貂脑冠状切片，开展关键神经祖细胞与神经元标志物的免疫荧光标记（immunofluorescent labelling）实验。所选用的标志物包括PAX6、SOX2、TBR2、HOPX、CPLX3、CTIP2、SATB2、TUJ1及DCX。通过半定量分析，描述各标志物在沿吻尾轴划分的限定皮层区域内的时空分布特征。

研究结果
早期放射状胶质细胞标志物PAX6与SOX2在胚胎期的脑室区（ventricular zone, VZ）中大量表达，随着出生后室下区（subventricular zone, SVZ）的扩张，其表达水平逐渐下降。由TBR2标记的中间祖细胞在产前的室下区内丰度较高，出生后则显著减少。HOPX可标记外放射状胶质细胞群体，这类细胞呈现独特的时空分布特征，在出生后阶段的皮层下板（subplate, SP）与皮层板（cortical plate）中的占比逐渐升高。CPLX3的表达始于出生后，可勾勒出成熟的皮层下板神经元，且其成熟过程呈现区域特异性模式。深层与表层神经元标志物CTIP2与SATB2呈现有序的层状表达时序，提示皮层层状结构的渐进式形成。通用神经元标志物TUJ1与DCX可体现有丝分裂后神经元的成熟与迁移过程，其时空梯度反映了不同区域的皮层分化进程。

讨论
本研究的详细图谱填补了雪貂组织学记录中的关键空白，可为利用雪貂模型开展哺乳动物皮层发生相关研究提供宝贵的参考资源。通过整合半定量评估与定性分析，本数据集为构建雪貂脑发育详细图谱的现有工作提供了助力。上述研究结果有望提升雪貂模型在神经发育研究中的应用价值，尤其是在涉及人类皮层畸形的转化研究场景中。