Investigating the Influence of Hormone Signaling Pathways on Mammary Gland Development and the Onset of Breast Cancer

January 21, 2016

Over their entire lifetime, 1 out of 8 women can suffer from breast cancer. The risk of developing breast cancer (mammary carcinogenesis) increases with a woman's age and is related to her reproductive history. The chance of mammary carcinogenesis is less for women who give birth to a child before the age of 30. However, it is now known that the risk of breast cancer also can depend on the number of times a woman experiences elevated blood serum progesterone levels, in relation to the menstrual cycle, before her first pregnancy. The female hormones estrogens, progesterone (known as the pregnancy hormone), and prolactin control breast development and are also shown to have an important role in mammary carcinogenesis. Unfortunately, the mechanisms underlying the roles of these hormones and other signaling proteins in carcinogenesis are still poorly understood. Here recent discoveries on the mechanism of how progesterone and its downstream signaling protein Wnt4, which promotes mammary gland development, control mammary stem cell function is reported. The progesterone/Wnt4 signaling pathway occurring in the breast (mammary gland) could provide explanations to the cancer promoting effects related to the frequency of serum progesterone peaks during menstrual cycles, use of oral contraceptive methods, and undergoing combined hormone replacement therapy.

Introduction

In order to overcome breast cancer and prevent its onset (mammary carcinogenesis), it is vital to better understand the biological mechanisms of breast development. It is well known that the risk of mammary carcinogenesis depends on a woman’s age and reproductive history [1]. However, it has been shown more recently that recurrent peaks in blood serum progesterone levels linked to the menstrual cycle is also an important risk factor for mammary carcinogenesis [23]. Normally, during a woman’s menstrual cycle (3–5 weeks for majority of women), the serum progesterone level is low during the first 1.5–2 weeks of the cycle (follicular phase), starts to increase with ovulation and reaches a peak several days afterwards (luteal phase), and then gradually drops back to low values by the end of the cycle (Figure 1) [4]. It is considered abnormal if a woman’s menstrual cycle is less than 3 weeks, or if she has elevated levels of serum progesterone outside of the last 1.5–2 weeks of the menstrual cycle whenever she is not pregnant [45].

Fig. 1: Chart of progesterone levels during a woman's menstrual cycle [4].

Mammary gland development

In order to better study mammary development mechanisms in more detail, the mouse is often used as a model organism. There are significant anatomical, histological, and hormonal similarities between the mouse mammary gland and human breast, which justify the use of a mouse as a model organism [6]. Additionally, there are practical reasons: the mouse mammary glands are amenable to in vivo and in vitro manipulations and techniques used in developmental biology and cancer research and the animal has a short life-cycle.

Mammary gland development occurs over multiple stages (Figure 2) [6]. At birth, only a rudimentary milk duct tree is present. Hormones induce communication between the mammary epithelial [7] and mesenchymal [8] cells and regulate their development. During puberty, this regulation leads to proliferation of mammary terminal end bud (TEB) epithelial cells. These cells grow into the mesenchymal layer eventually reaching the fat pad at which point they begin to form 2nd and 3rd degree branching.

Fig. 2: Schematic representation of postnatal mouse mammary gland development showing the various stages: puberty; estrus (female sexually receptive); pregnancy; lactation (milk secretion); and involution (cessation of milk secretion).

The mammary duct is composed of inner luminal epithelial layer and outer myoepithelial layer. The two major ovarian hormones, i.e., 17-β-estradiol (E2, estrogen family) [9] and progesterone (P) [10], are involved in postnatal (after birth) mammary gland development. These hormones also are known to be factors in mammary carcinogenesis. The signaling from these hormones for breast development takes place via nuclear receptors [11], the estrogen receptor α (ERα) and  the progesterone receptor (PR), respectively, expressed in a subset of epithelial cells in the luminal [12] layer.

Among mammary epithelial cells positive for CD24 [13], populations with high surface expression of integrin β1 (CD29hi[14] or integrin α6 (CD49fhi[14] were more often able to produce new milk ducts in mammary fat pads from which the endogenous epithelium was surgically removed. These epithelial cells are considered bipotent mammary stem cells [15] which yield both luminal and basal/myoepithelial [16] cells [17]. However, the relevance of the dramatic growth of these bipotent stem cells in response to hormones is questioned by lineage-tracing experiments showing that postnatal mammary gland development is largely driven by luminal and basal/myoepithelial lineage-restricted stem cells.

The fact remains that most cell proliferation occurs in the luminal compartment and the majority of breast cancers arise from luminal and/or luminal progenitor [18] cells. Repeated exposure to elevated serum progesterone levels during menstrual cycles is associated with increased risk for breast carcinogenesis. This observation begs the question how important progesterone is to mammary stem cell function in intact tissue and which signaling pathways it uses to influence the various types of stem cells.

Involvement of the progesterone/Wnt4 signaling pathway in mammary gland development and regeneration

It has been demonstrated that mammary epithelial tissue can be serially engrafted to cleared mammary fat pads for up to seven generations [17]. As an assay [19], grafted epithelium preserves the epithelial architecture with its associated extracellular matrix (ECM) [8], fibroblasts [8], and immune cells. At present, the surgical engraftment (serial transplantation) of mammary epithelial tissue is the sole way to assay comprehensively the ability of various types of stem and progenitor cells to regenerate mammary tissue. This assay has been combined with different genetic mutant strains to define the relative contributions of progesterone signaling and its downstream mediators Wnt4 and RANKL [20] to determine the regenerative potential of the mammary epithelial cells.

The results reported here address recent findings concerning the impact that the ovarian hormone progesterone and signaling protein Wnt4 might have on mammary carcinogenesis mechanisms.

Methods

Mice

All mice were maintained and handled in accordance with Swiss guidelines for animal safety. All the animal experiments were approved by the ethic veterinary committee of canton Vaud, Switzerland. The mice for this study were kept with mixed genetic background (mouse strains 129SV and C57BL6) [17].

Image analysis and staining

The outgrowth of mammary ducts expressing EGFP [21] (for serial transplantations) or dTomato [21] was visualized by fluorescence stereo microscopy [17]. After mammary gland whole-mounting and X-gal or carmine alum staining, images were acquired using a Leica MZ FLIII, Leica M205FA, or Leica MZ16F fluorescence stereo microscope. The area of the fat pad filled by the mammary ducts was quantified using axiovison rel 4.7 software. For immunostaining, mammary glands were fixed in paraformaldehyde and embedded in paraffin wax. Sections 5 μm thick were cut and immunohistochemical or immunofluorescence staining were performed [17]. Images were acquired on a Leica DM2000 or Zeiss Axioplan fluorescence compound microscope.

Isolation of RNA and semi-quantitative qPCR

Mammary glands were homogenized in TRIzol® (Invitrogen) [17]. Total RNA [22] was isolated from fragments using RNeasy (Quiagen). cDNA [23] was synthesized using random p(dN)6 primers [24] and MMLV reverse transcriptase [25]. Semi-quantitative, real-time PCR (qPCR) [26] analysis was performed in triplicate with a series of primer sequences [17].

Statistical data analysis

Data sets were compared using the 2-tailed, paired Student's t-test method in order to determine significant difference [17]. For each data set, a mean and standard deviation (σ, SD) value are reported. The percent extent of fat pad filling for each generation was compared between contralateral outgrowths by the Wilcoxon signed rank test. A p-value < 0.05 indicated a statistically significant difference between data sets.

Results

Role of progesterone receptor (PR), RANKL, and Wnt4 in renewal of mammary epithelium

To assess the role of PR, RANKL, and Wnt4 signaling in mammary stem cell control, pieces of intact mammary epithelium were serially engrafted from the following mice littermates:

  1. PR: wild type [27] (WT; homozygous [28] PR positive, PR+/+) and mutant [27] (MT; homozygous PR negative, PR/–);
  2. RANKL: wildtype (RANKL+/+) and mutant (RANKL/–); and
  3. Wnt4: wildtype (Wnt4+/+) and mutant (Wnt4/– or Wnt4+/–).

The intact epithelial fragments were grafted into contralateral (side-by-side) mammary fat pads. The fat pads were surgically cleared of their endogenous epithelia (Figure 3A) prior to grafting. Wnt4/– mice die shortly after birth and, to overcome this problem, mammary epithelial buds harvested from mouse embryonic day 12.5/13.5 were transplanted. To easily distinguish the engrafted cells from the endogenous epithelium that may inadvertently be left after surgery, donors that expressed EGFP [21] were used. The extent of outgrowth in the engrafted mammary glands was determined after 8–12 weeks. Mammary outgrowth resulting from the contralateral PR/–, RANKL/–,  Wnt4/–, Wnt4+/–, and WT (+/+) grafts were dissected and re-transplanted (Figure 3A).

As expected, the PR+/+ epithelia completely reconstituted most of the fat pads over 4 serial transplant generations, but the PR/– epithelia ceased to reconstitute the mammary gland by the 3rd cycle (Figure 3B). The RANKL+/+ epithelia also fully reconstituted fat pads in most hosts over 4  generations of serial transplants (Figure 3C). RANKL/– epithelia had the same regeneration capacity as RANKL+/+ with one significant difference being that the MT grafts generated fewer side branches [17]. The mammary outgrowth derived  from the Wnt4+/+ and the  Wnt4+/ mammary buds reconstituted the mammary gland to the same extent as the WT epithelia derived from postnatal mammary glands through 3 cycles (Fig. 3D). However, the Wnt4/– epithelia only established 50% of the fat pad in the 1st cycle and only 10 % by the 3rd cycle. Fat pad reconstitution is more significantly reduced with Wnt4/– epithelial grafts than with PR/– grafts (comparison between contralateral controls, Figure 3E). This result indicates that Wnt4 has an important role in mammary stem cell function and that PR does not exclusively control Wnt4 expression. RANKL appears to have no essential control of mammary stem cell function [17].

The box plot below (Figure 3E) shows the difference in percentage of fat pad reconstitution between WT and MT contralateral grafts (% from WT – % from MT; MT denoted as knockout [29] {KO}) for each generation.

Fig. 3A: Experimental scheme for the mammary gland development study. Mammary tissue fragments dissected from wild type (WT) or mutant (MT) donor mice were engrafted to contralateral mammary fat pads of recipient mice whose endogenous epithelia were surgically ablated beforehand. The engrafted glands were assessed by fluorescence stereo microscopy 8–12 weeks later. Then new epithelial fragments were dissected for serial engraftment.

Fig. 3B: Serial transplantations of PR+/+ and PR–/– mammary epithelia. Fluorescence stereo micrographs of 3rd generation mammary outgrowths from 8-week-old PR+/+; EGFP and PR–/–; EGFP donor mice. Scale bar = 200 μm.

Fig. 3C: Serial transplantation of RANKL+/+ and RANKL–/– mammary epithelia. Fluorescence stereo micrographs of 3rd generation mammary outgrowths from 5-week-old RANKL+/+; EGFP and RANKL–/–; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL–/–; EGFP epithelium. Scale bar = 200 μm.

Fig. 3D: Serial transplantation of Wnt4+/+ and Wnt4–/– mammary epithelia. Fluorescence stereo micrographs of 3rd generation mammary outgrowths derived from mammary buds of Wnt4+/+; EGFP and Wnt4–/–; EGFP embryos. Scale bar = 200 μm.

Fig. 3E: Box plot showing the difference in percent of fat pad filling (reconstitution) between the wildtype (WT) and mutant (KO or MT) contralateral grafts (WT – KO) in each transplant generation. The p-values were determined by the Mann–Whitney U-test.

Role of progesterone and estrogens in Wnt4 expression

The results mentioned in the section above, that Wnt4 influences mammary regeneration potential in a more significant way than PR signaling, suggests that Wnt4 may have functions in stem cell stimulation independent of PR.

Mouse mammary glands (Wnt4::EGFP) having WT, ERα/–, and PR/– genetic backgrounds were analyzed by fluorescence stereo microscopy. Ductal outgrowth was compared using the red fluorescent marker dTomato. Neither ERα nor PR deletion altered Wnt4::EGFP expression indicating that perinatal [30] Wnt4 expression is largely independent of ERα and PR signaling [17].

To determine the roles of the 2 major ovarian hormones, progesterone (P) and E2 (17-β-estradiol of the estrogen family [9]), in Wnt4 expression, first epithelial enriched organoids freshly isolated from mammary glands of pubertal (6 weeks old) and adult (11 weeks old) mice were tested for progesterone- and E2-induced Wnt4 mRNA [31] expression. Wnt4 mRNA expression was approximately 9x increased for pubertal mice versus 5x for adult mice by progesterone exposure, whereas E2 elicited only about a 2x increase of Wnt4 mRNA in pubertal mice (Figure 4A). To assess the physiological importance of PR signaling for pubertal Wnt4 expression, Wnt4::GFP epithelium derived from PR/– or PR+/+ (WT) donors was grafted to contralateral, cleared fat pads. The engrafted glands were analyzed when the recipients were in the puberty stage. Fluorescence stereo microscopy (dTomato) data reveal non-recombined cells, confirming the presence of ductal outgrowth in the PR/– and PR+/+ grafts (Figure 4B–C). EGFP expression was readily detected in the PR+/+ graft (Figure 4D), but absent/not present in the PR/– grafts (Figure 4E). Double (dTomato and EGFP) fluorescence stereo microscopy on a PR+/+ control graft shows EGFP present in the TEBs (Figure 4F,H). In the contralateral PR/– grafts, some EGFP expression is observed at the starting point of the outgrowth (Figure 4G). These results indicate that pubertal Wnt4 expression is mediated by PR signaling [17].

Fig. 4: A) Bar plots showing normalized values of relative Wnt4 mRNA expression in mammary organoids from pubertal and adult mice; C is the vehicle control, E2 is 17-β-estradiol exposure, and P is progesterone exposure. The error bars represent the mean value ± σ (SD). B–H) Epifluorescence stereo micrographs of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium: PR+/+ (B, D, F, H) or PR/– (C, E, G). dTomato expression (B, C); EGFP expression (D, E) double (dTomato and EGFP) epifluorescence (F, G, H) on contralateral engrafted glands. Arrowheads point to TEBs (F, H) or the central growth node/site of primary mammary outgrowth (G). Scale bar = 5 mm (B–G), 1 mm (H).

Mammary myoepithelial cells activation by canonical Wnt signaling

The peak in myoepithelial β-galactosidase [32] activity during mid-pregnancy suggests that serum progesterone levels and Wnt4 expression may induce activation of canonical Wnt signaling [33]. Mammary glands analyzed during progesterone-low estrus [34] had lower β-galactosidase activity (Figure 5A) and lower Wnt4 and Axin2 [35] mRNA levels (Figure 5B) than those analyzed during progesterone-high diestrus phase [34]. To determine if progesterone signaling induces canonical Wnt signaling, ovariectomized (ovaries removed) Axin2::LacZ [36] females were treated with the vehicle, E2 alone and E2, to restore PR expression, and progesterone (E+P). Axin2 transcription, evidenced by β-galactosidase activity, was stimulated by the E+P combination, but not by the vehicle or E2 alone (Figure 5C). Thus, progesterone stimulation results in increased transcription of Axin2. Multiple Wnts, some of which are secreted by mammary stromal cells, have been implicated in canonical Wnt signaling activation [17]. To assess whether canonical Wnt signaling requires Wnt4 expression, Axin2::LacZ transgenic mice generated in a Wnt4/– background were exploited. β-galactosidase activity was readily detected after 8 days of pregnancy in Wnt4+/+ grafts from mice engrafted with mammary buds from the Axin2::LacZ+ embryos, but abrogated in the contralateral Wnt4/– Axin2::LacZ+ mammary bud derived out growth (Figure 5D). Similarly, canonical Wnt signaling activation was diminished in PR+/ and abrogated in PR/– epithelia (Figure 5E), indicating that both PR and Wnt4 are required for canonical Wnt signaling activation in the myoepithelium.

A
B
C
D
E
 

Fig. 5: A) Representative whole-mount stereo micrographs of X-gal [36] (blue)- and carmine alum (magenta)-stained mammary gland biopsies from Axin2::LacZ female mice in estrus and diestrus. B) Ratio of diestrus/estrus relative Wnt4 and Axin2 mRNA expression in mammary glands from mice in estrus versus diestrus assessed by normalized semi-quantitative qRT–PCR. The blue line represents the mean value. C) Stereo micrographs of X-gal- and carmine alum-stained mammary glands from ovariectomized Axin2::LacZ females: treated for 72 hours with control (left), 17-β-estradiol (E2) (center), E2 and progesterone (P) (right). D–E) Stereo micrographs of contralateral glands whole-mounted and X-gal "stained" after engraftment with mammary buds from Axin2::LacZ transgenic mice and Wnt4+/+ or Wnt4/– female embryos (D) or Axin2::LacZ transgenic mice and either PR+/ or PR/– female mice (E). After 8–9 days of pregnancy, β-galactosidase expression reflecting Axin2 transcription is readily detected in PR+/ and Wnt4+/+ mammary epithelia, but not in the PR/– and Wnt4/– counterparts. Blue is X-gal staining and magenta is carmine alum staining. All scale bars = 200 μm.

Discussion

The results of this study show that early (perinatal [30]) Wnt4 expression is functionally important [17]. Further results indicated that canonical Wnt signaling in the myoepithelium requires PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4 [17]. In addition, data depict Wnt4 as a pivotal control factor of stem cell function for postnatal mammary gland development and that it has a novel role in perinatal and pubertal development in conjunction with progesterone.

Although previous hormone stimulation experiments had shown that estrogens induce Wnt4 expression, this study finds that genetic deletion of PR signaling completely abrogated Wnt4 expression during puberty [17]. Still, the two ovarian hormones remain intertwined in Wnt4 control with ERα signaling acting indirectly as an upstream regulator of PR expression (Figure 6A) [17].

The study shows RANKL is not important for stem cell potential, which is in contradiction with previous results based on assays with dissociated cells [17]. Compared to single-cell-based assays in which a defined number of cells are injected, grafting of intact epithelial fragments does not allow one to determine the fraction of cells endowed with regenerative potential. However, unlike dissociated cells, the method can give a semi-quantitative appreciation of an intact regeneration potential in a physiological tissue context. In fact, it is well known that an intact microenvironment is important to stem cell function.

The activation of canonical Wnt signaling when PR+ luminal cells secrete Wnt4 implies that the myoepithelial/basal cells are a central component of the microenvironment that controls different types of stem cells (Figure 6B) [17]. The myoepithelial/basal cells are ultimately under control of progesterone and Wnt4, thus, stem cell activity is linked to reproductive needs.

The study also finds that myoepithelial/basal cells are Wnt4’s prime target [17]. Wnt1 is known to be an oncogene [37] in the mouse mammary gland and Wnt signaling is key for its development. However, no mutations in the intracellular Wnt signaling components have been found in breast carcinomas. Wnt signaling activation in the myoepithelial cells may indirectly promote tumorigenesis via gene expression changes that induce secretion of stimulatory signals and/or modulation of the extracellular matrix leading to activation of the luminal progenitor cells. Luminal progenitor cells with existing oncogenic mutations could further expand in response to Wnt4 stimulation of the myoepithelium (Figure 6C) [17]. In parallel, Wnt4 acts on bipotent mammary stem cells to increase their numbers. As a consequence, the luminal progenitor cells are amplified which are also more prone to oncogenic attack than the more differentiated luminal epithelial cells.

Fig. 6: A) Model for Wnt4 expression during mammary gland development. Schematic representation of gland development (bottom) and control of mammary stem/progenitor cells by hormones (top). Wnt4 is important for stem cell activation throughout postnatal development. PR signaling is required for Wnt4 expression during puberty and adulthood. ERα signaling induces PR expression. B) Model of Wnt4 action in the mammary epithelium. Progesterone stimulates Wnt4 induction in the PR+ luminal cells (LC). The secreted Wnt4 acts on the adjacent basal/myoepithelial cells (MC). Wnt4 activates canonical Wnt signaling in the myoepithelial cells which causes changes in gene expression. This signaling results in the secretion of factors inducing changes in the ECM [8] that in turn impinge on stem cells (SC), luminal-restricted stem cells (L-RSC), and basal-restricted stem cells (B-RSC). C) Model for the tumorigenic effects of progesterone and Wnt4 in the mammary epithelium. Repeated activation of this intercellular signaling cascade, downstream from PR signaling, may promote tumorigenesis by expanding the stem/progenitor cell compartment, as well as, the luminal progenitor cells with oncogenic mutations.

Conclusions

It is known that the progesterone/Wnt4 signaling pathway operates also in the human breast [17], thus results reported here very likely have implications for mammary carcinogenesis in women. The activation of the progesterone/Wnt4 pathway may promote the tumorigenesis effects in correlation with: the frequency of blood serum progesterone peaks during menstrual cycles; oral contraception use; and experiencing combined hormone replacement therapy with progestins. Specific progesterone receptor modulators and Wnt inhibitors, alone or together with RANKL inhibitors, may be effective in breast cancer prevention or treatment. Their use may be especially helpful as preventive strategies for pre-menopausal women at high risk of developing breast cancer.

References/Notes

  1. Breast Cancer: Know the Risks Infographic. Division of Cancer Prevention and Control, USA Centers for Disease Control and Prevention.
  2. Brisken C: Progesterone signalling in breast cancer: a neglected hormone coming into the limelight. Nature Reviews Cancer 13: 385–96. doi: 10.1038/nrc3518.
  3. Kuhl H, Schneider HPG: Progesterone – promoter or inhibitor of breast cancer. Climacteric 16, Suppl. 1 (2013). doi: 10.3109/13697137.2013.768806.
  4. Häggström M: Reference ranges for estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone during the menstrual cycle. Wikiversity Journal of Medicine 1 (2014). doi: 10.15347/wjm/2014.001, ISSN: 20018762.
  5. Walker HK, Hall WD, and Hurst JW:Long WN: Clinical Methods – The History, Physical, and Laboratory Examinations; in Long WN: Abnormal Vaginal Bleeding, Ch. 173, 3rd Ed.. Butterworths, Boston (1990). ISBN-10: 0-409-90077-X.
  6. Macias H, Hinck L: Mammary Gland Development. Wiley Interdiscip. Rev. Dev. Biol. 1 (4): 533–57 (2012). doi: 10.1002/wdev.35.
  7. Epithelial cells comprise the tissue linings of cavities and surfaces of blood vessels and organs throughout an animal's body. Epithelium is one of the four basic animal tissues.
  8. Mesenchymal cells lack polarity (spatial differences in shape, structure, and function), are surrounded by a large extracellular matrix (ECM; the ensemble of extracellular molecules that provides structural and biochemical support to the surrounding cells), and develop into connective tissue or tissues of the lymphatic and circulatory systems. Fibroblast cells are derived from mesenchymal cells. Fibroblasts synthesize the ECM and collagen, the stroma (tissue structure framework), and participate in tissue healing.
  9. 17-β-estradiol, a steroid, is the most prevalent endogenous sex hormone of the estrogen family.
  10. Progesterone, also called pregn-4-ene-3,20-dione, a steroid, is an endogenous progestogen sex hormone.
  11. Nuclear receptors are a class of proteins within cells that specialize in the sensing of steroid and thyroid hormones and certain other molecules. They also work in conjunction with other proteins to regulate gene expression in relation to an organism’s development, homeostasis, and metabolism.
  12. Luminal derives from lumen, the inside space of a tubular structure, such as an artery or intestine, or a cellular component, such as, the endoplasmic reticulum.
  13. CD24 is a cell adhesion molecule (CAM), a glycoprotein expressed at the surface of most B lymphocytes (white blood cells) and differentiating neuroblasts (cells developing into neurons).
  14. Integrin β1 (CD29) and α6 (CD49f) are proteins that are integrin units. Integrin proteins are membrane receptors involved in cell adhesion and recognition.
  15. Bipotent stem cells are undifferentiated cells which can differentiate into 2 types of specialized cells and reproduce to make more stem cells.
  16. Myoepithelial cells are usually found in glandular epithelium as a thin layer above the basement membrane, but generally beneath the luminal cells.
  17. Rajaram RD, Buric D, Caikovski M, Ayyanan A, Rougemont J, Shan J, Vainio SJ, Yalcin-Ozuysal O, and Brisken C: Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk. EMBO online (20 Jan 2015). doi: 10.15252/embj.201490434.
  18. Progenitor cells are similar to stem cells, but can only differentiate into specific types of cells  Whereas stem cells can replicate indefinitely, progenitor cells can divide only a limited number of times.
  19. An assay is an analytical procedure to qualitatively or quantitatively assess the presence, amount, or functional activity of a target entity, often called the analyte.
  20. RANKL stands for Receptor Activator of Nuclear factor Kappa-B Ligand, also known as TNFSF11 (Tumor Necrosis Factor ligand Super Family member 11) and ODF (Osteoclast Differentiation Factor). RANKL is a membrane protein known to affect the immune system and control bone regeneration.
  21. EGFP stands for Enhanced Green Fluorescence Protein, a protein that emits green when excited by blue light. In cell and molecular biology, the EGFP gene is frequently used as a marker of protein expression. dTomato, a dimer of the DsRed fluorescent protein, emits red when excited by green light.
  22. RNA stands for Ribonucleic Acid, a polymeric molecule which takes part in coding, decoding, regulation, and expression of genes. The nucleic acids RNA and DNA (Deoxyribonucleic Acid), along with proteins and carbohydrates, constitute the three major macromolecules essential for life.
  23. cDNA (complementary DNA) is double-stranded DNA synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is also used to express a specific protein in a cell that does not normally express that protein.
  24. Primers are strands of short nucleic acid sequences (approximately 10 base pairs) that serve as starting points for DNA synthesis in the polymerase chain reaction (PCR) method.
  25. MMLV (Moloney Murine Leukemia Virus) reverse transcriptase is an RNA-directed DNA polymerase. The enzyme can synthesize a cDNA strand from a primer using either RNA or single-stranded DNA as a template.
  26. qPCR refers to real-time Polymerase Chain Reaction, a molecular biology technique used for the amplification of a targeted DNA molecule.
  27. Wild type (WT) refers to the usual observable characteristics or traits (phenotype) of the typical form of a species as it is found in nature (no artificially induced gene mutations). Mutant (MT) refers to an organism or new genotype (genetic characteristics) resulting from mutation, i.e., a base pair sequence change within the DNA of the organism’s genes. Mutations can occur both naturally or be induced artificially.
  28. A diploid (matching pair of chromosomes) organism with the same alleles (alternative forms of the same gene) is called homozygous, but if the alleles are different, then it is called heterozygous.
  29. A knockout (KO) is a genetic technique in which one of an organism's genes is made inactive, thus it is "knocked out".
  30. Perinatal refers to the period just before and after birth.
  31. Messenger RNA (mRNA) is a ribonucleic acid molecule that conveys genetic information from DNA to the ribosome, a cell organelle where proteins are synthesized. mRNA specifies the amino acid sequence for the protein products resulting from gene expression.
  32. Galactosidases (glycoside hydrolases) are enzyme catalysts that hydrolyze galactosides (glycoside with galactose) into monosaccharides (simple sugars). There is the α-galactoside and β-galactosidase form.
  33. There are 3 Wnt protein signaling pathways: the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt/calcium pathway.
  34. The estrous cycle refers to recurring physiological changes that occur in mammalian therian (give birth to live young, no egg) females due to reproductive hormones. It starts after sexual maturity is interrupted by anestrous phases (sexual cycle pauses due to seasonal changes and reduced light exposure) or pregnancies. There are typically 4 phases of the estrous cycle: proestrus (follicles of the ovary start to grow and estrogen causes the endometrium [uterus lining] to develop), estrus (the female is sexually receptive or "in heat"), diestrus or metestrus (corpus luteum gland is active and produces progesterone and estrogen).
  35. Axin2 is one of the Axin proteins which presumably play a role in the regulation of b-catenin stability. The protein b-catenin is a subunit of the cadherin protein complex and acts as an intracellular signal transducer in the Wnt signaling pathway.
  36. A fragment of the lacZ gene (lacZα) and a mutant lacZ gene (lacZΔM15) are complementary. When the genes are expressed together, as when a plasmid containing lacZα is transfected into a lacZΔM15 containing cell, the enzyme β-galactosidase is formed. The presence of active β-galactosidase is detected by exposure to X-gal resulting in a blue-coloration of the cells. X-gal is an analog of lactose and can be hydrolyzed by β-galactosidase. The blue/white screening method exploits the complementation of lacZα and lacZΔM15 and the hydrolysis of X-gal by β-galactosidase for cell identification.
  37. An oncogene is a gene that can provoke the onset of cancer. Tumor cells often have oncogenes expressed at high levels.

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