Expression and modulation of <i>Gli1-LacZ</i> in the adult nipple during the pregnancy cycle.

<p>Gli1-LacZ expression is visible at the neck of the mammary sprout (red arrow) in skin whole-mounts of E18.5 <i>Gli1^lzki/+</i> embryos at low (A) and higher power (A’). Histological section through the sprout shows expression of Gli1-LacZ within the stroma (red arrow) surrounding the sprout (black arrow) underneath the nipple sheath (black arrowheads) at low (B) and higher power (B’). <i>Gli1-lacZ</i> is expressed within the dermal component but not the epithelium in virgin (C), mid pregnant (D), lactating (E) and involuting (F) nipples. Immunohistochemistry for desmin (G, J), SMA (H, K) and vimentin (I, L) on serial sections of a 15.5 day pregnant nipple demonstrated that <i>Gli1-lacZ</i> was expressed in both smooth muscle cells and fibroblasts but not myofibroblasts. <i>Gli1-lacZ</i> was also found near and surrounding peripherin positive nerve tracts (M) and both Von Willebrand positive vessels (N).</p

Percentages of MRs showing phenotypic abnormalities in female <i>Gli2^1ki/1ki; Gli3^xt/+</i> embryos at E18.5.

<p>Numbers represent percentages of MRs showing loss, evagination or impairment in invagination from a total ‘n’. Abbreviations: Imp. Invag: Impaired Invagination; n: total number of MRs analyzed.</p

Percentages of evaginated MRs in male <i>Gli3^xt/xt</i> embryos at E16.5 and E18.5.

<p>Numbers represent percentages of MRs that protrude from the surface of male skin whole-mounts from a total ‘n’. Abbreviations: E: embryonic day; n: total number of putative sites for MRs on male skin whole mounts.</p

Percentages of MRs showing retention in male <i>Gli3^xt/xt</i> embryos at E16.5 and E18.5.

<p>Numbers represent percentages of MRs retained on male skin whole-mounts from a total ‘n’. Abbreviations: E: embryonic day; n: total number of putative sites for MRs on male skin whole mounts.</p

Misactivation of Hh signaling detrimentally affects MR invagination and hair follicle suppression in females and eradication of MRs in males.

<p>X-Gal stained whole-mounts (A–E) and sections (F-H) of <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos were examined at E18.5. In whole-mounts of female skins, MR#2 protruded prominently and showed encroachment of hair follicles inappropriately close to the evaginated MR (A). Examination of male skin whole-mounts revealed retention of MR#1, #2 and #4 at low (B) and high power (C, D, E respectively), similar to that seen in <i>Gli3^xt/xt;Gli1^lzki/+</i> embryos. Serial sections through a male MR#2 from <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos showed Gli1-LacZ-positive hair follicles close to the protruding bud (F; NFR counterstain) and weak expression of Tenascin C (G) and loss of AR (H) by immunohistochemistry.</p

Percentages of MRs showing retention and evagination in male <i>Gli2^1ki/1ki; Gli3^xt/+</i> embryos at E18.5.

<p>Numbers represent percentages of MRs that are retained or protrude [in brackets] from the surface of male skin whole-mounts from a total ‘n’. Abbreviations: n: total number of putative sites for MRs on male skin whole mounts.</p

<i>Tbx3</i> mRNA expression in <i>Gli3^xt/xt</i> embryos.

<p>Whole-mount <i>in situ</i> hybridization for <i>Tbx3</i> in wt (A, B) and <i>Gli3^xt/xt</i> (C, D) embryos. At the 40-somite stage, wt embryos (A) express <i>Tbx3</i> mRNA in the mesenchyme between the fore- and hind-limb. This band of expression is narrower in <i>Gli3^xt/xt</i> embryos at this stage (yellow bracket). At the 45-somite stage <i>Tbx3</i> is reduced within the mesenchyme but is induced within the epithelium of developing mammary placodes #3 (white arrow) in wt embryos (B). <i>Gli3^xt/xt</i> embryos (D), show a narrower band of <i>Tbx3</i> expression between the fore- and hind-limb and fail to concentrate epithelial expression within placode #3 (white arrow). The control <i>in situ</i> hybridization for <i>Tbx3</i> sense probe is shown in (E). Abbreviations: F – fore-limb, H – hind-limb, s – somite.</p

Percentages of MRs showing phenotypic abnormalities in female <i>Gli3^xt/xt</i> embryos at E18.5.

<p>Numbers represent percentages of MRs showing loss, evagination or impairment in invagination from a total ‘n’. Abbreviations: Imp. Invag: Impaired Invagination; MR: mammary rudiment; xt: extra toe mutation; n: total number of MRs analyzed.</p

Failure of MR#2 invagination in <i>Gli3^xt/xt</i> embryos.

<p>Analysis of the inner surface of skin whole-mounts from E18.5 wt (A, B, C) and <i>Gli1^lzki/+</i> (D, E, F) and <i>Gli3^xt/xt; Gli1^lzki/+</i> (G, H, I) embryos stained with X-Gal to detect <i>Gli1-LacZ</i> reporter expression (blue) within hair follicles and counterstained with carmine alum (pink). MRs#1 (A, D, G) and #4 (C, F, I) show comparable development at E18.5 in all three genotypes: sprouts are clearly visible (arrow). In control wt (B) and <i>Gli1^lzki/+</i> (E) embryos sprout #2 has elongated and branched several times but in <i>Gli3^xt/xt; Gli1^lzki/+</i> embryos (H), MR#2 shows no evidence of sprouting towards the fatpad.</p

<i>Bmp4</i> mRNA expression is displaced in <i>Gli3^xt/xt</i> embryos.

<p>Whole-mount <i>in situ</i> hybridization for <i>Bmp4</i> in wt (A, B) and <i>Gli3^xt/xt</i> (D, E) embryos. In 40-somite stage wt embryos (A) <i>Bmp4</i> is expressed ventrally. This expression is more intense in <i>Gli3^xt/xt</i> embryos at this stage (D). At the 45-somite stage, expression is reduced in wt embryos (B arrow), but expression is displaced dorsally and centrally into the locale of the developing mammary placode #3 in <i>Gli3^xt/xt</i> embryos (E arrow). Distance from the axilla to the most distal point of <i>Bmp4</i> expression (B, E white dotted lines) was measured in mm. Sections through a 45-somite wt embryo (C) and higher power (C’) show mesenchymal expression ventral to the developing placode #3 (arrow). Sections through a 45-somite <i>Gli3^xt/xt</i> embryo (F) and higher power (F’) show the displaced <i>Bmp4</i> expression. Control <i>in situ</i> hybridization with <i>Bmp4</i> sense probe is shown on a wt embryo in (G). Abbreviations: F – fore-limb, H – hind-limb, nt – neural tube, s – somite.</p