Lineage tracing for mammary stem cells using Hopx reporter mice

doi:10.12307/2025.088

Abstract

Abstract: BACKGROUND: Mammary stem cells are vital for the development and homeostasis of mammary gland tissue. The occurrence of breast cancer has a close relationship with the mammary stem cells. Recent studies have shown that Hopx, as an important transcriptional regulator of morphogenesis and cell differentiation, has been confirmed to be expressed in a variety of adult stem cells such as nerves, intestines, hair follicles and lungs. However, its role in mammary stem cells has not been reported so far.
OBJECTIVE: To investigate whether Hopx expression marks mammary stem cells.
METHODS: (1) Female Hopx-LacZ transgenic mice aged 8 weeks were selected to detect the background expression of Hopx in breast tissue by β-galactosidase staining. (2) Female wild-type mice at 4, 6, and 8 weeks of age and 14.5 days of gestation were selected for whole-tissue magenta staining and K14 and K8 immunofluorescence staining, respectively. (3) Female Hopx-CreERT2; Rosa26LacZ transgenic mice aged 8 weeks and 17.5 days of gestation were selected and stained with breast β-galactosidase. (4) The 4-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times), and breast β-galactosidase staining was performed 4 weeks after injection. The 8-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times), and breast β-galactosidase staining was performed 4 and 10 weeks after the last injection. (5) Female Hopx-CreERT2; Rosa26LacZ transgenic mice aged 8 weeks were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times). Hopx-CreERT2;Rosa26LacZ transgenic mice were pregnant 2 weeks after injection. The mammary tissue of mice at 17.5 days of the first pregnancy and 17.5 days of the third pregnancy was stained with β-galactosidase.
RESULTS AND CONCLUSION: (1) The results of β-galactosidase staining showed that the mammary ducts of Hopx-LacZ transgenic mice at 8 weeks of age did contain Hopx-positive cells and were located in the basal epithelia, with a small number. (2) Whole-mount staining of mammary glands and immunofluorescence staining results exhibited that the mammary glands of mice had different characteristics with corresponding developmental stages such as puberty, maturity, and pregnancy, and underwent a series of complex epithelial remodeling processes. (3) The results of β-galactosylase staining showed that Hopx-labeled positive cells in the mammary duct of Hopx-CreERT2; Rosa26LacZ transgenic mice at 17.5 days of gestation increased compared with female Hopx-CreERT2; Rosa26LacZ transgenic mice at 8 weeks of age. (4) The results of β-galactosylase staining showed that the Hopx-labeled positive cells in the mammary glands of 4- and 8-week-old female Hopx-CreERT2; Rosa26LacZ transgenic mice after tamoxifen injection were located in the basal epithelium with a small number. (5) The results of β-galactosidase staining showed that Hopx-labeled positive cells in the mammary glands of mice at 17.5 days of the first and third gestation were located in the basal epithelia around the alveoli, and the number of Hopx-labeled positive cells at 17.5 days of the third gestation was more. (6) In conclusion, Hopx reporter-marked basal epithelial cells belong to dormant mammary stem cells, which are responsible for the growth of the mammary glands during pregnancy and contribute to acinar formation.

Key words: Hopx, mammary stem cell, lineage tracing, gestational period, acinar formation

CLC Number:

Shi Jianyun, Li Wenjing, Peng Ying, Jia Zhenhua, Zhang Shujin, Tan Lulu, Yuan Yitong, Du Ruochen. Lineage tracing for mammary stem cells using Hopx reporter mice[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(13): 2720-2727.

Figures/Tables 3

2.1 不同发育时期的乳腺结构如图1A所示，青春期(4-6周龄)野生型小鼠的乳腺导管末端为膨大的末端乳芽结构，并且导管刚延伸至淋巴结附近并未布满整个脂肪垫；成熟期时(8周)，野生型小鼠的乳腺导管分支变多并出现大量次级分支，且并能够布满整个脂肪垫；妊娠期时，野生型小鼠乳腺进一步发育形成腺泡结构。K14标记的为基底上皮细胞，K8标记的为腔上皮细胞。实验进一步通过免疫荧光染色分析观察基底上皮细胞和腔上皮细胞分别在乳腺导管(来源于4周龄和8周龄小鼠)、末端乳芽结构(来源于6周龄小鼠)和腺泡结构(来源于妊娠期小鼠)中的分布情况，见图1B。综合以上结果表明，小鼠乳腺在青春期、成年期及妊娠期等不同发育阶段经历了一系列复杂的上皮重塑过程，并且具有各自相应时期依赖性的特征。 2.2 Hopx的表达标记了一小群乳腺干细胞 8周龄雌性Hopx-LacZ转基因小鼠腹股沟处第四对乳腺β-半乳糖苷酶染色结果显示，乳腺导管中确实含有Hopx阳性细胞(蓝色)且大多数位于基底上皮，但数量较少，见图2。采用雌性Hopx-creERT2;Rosa26LacZ转基因小鼠模型来示踪Hopx在乳腺组织中的表达情况，小鼠构建方式如图3A所示。8周龄雌性Hopx-creERT2;Rosa26LacZ转基因小鼠肠道β-半乳糖苷酶染色结果显示，肠道中出现了Hopx报告基因标记的细胞，见图3B，表明雌性Hopx报告基因小鼠可以作为有效的谱系追踪工具小鼠。8周龄及妊娠17.5 d的雌性Hopx-creERT2;Rosa26LacZ转基因小鼠乳腺β-半乳糖苷酶染色结果显示，妊娠期小鼠乳腺导管蓝色着色较深，表明Hopx阳性细胞数量较多，见图3C。以上结果表明Hopx标记了一种少见的乳腺上皮细胞群，并且可能在妊娠期时数量增多。"

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