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First published online 26 January 2006
doi: 10.1242/dev.02261

Development 133, 823-832 (2006)
Published by The Company of Biologists 2006
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Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis

Phyllis Strickland1,*, Grace C. Shin1,*, Andrew Plump2, Marc Tessier-Lavigne3 and Lindsay Hinck1,{dagger}

1 Department of Molecular, Cell and Developmental Biology University of California, Santa Cruz Santa Cruz, CA 95064, USA.
2 Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065, USA.
3 Genentech, Incorporated, 1 DNA Way, South San Francisco, CA 94080, USA.


Figure 1
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  		Fig. 1. Expression patterns of Slit2 and Slit3 during mammary gland development. (A) Schematic of an EB with subtending duct. (B,C) SLIT2 immunostaining on (B) +/+ and (C) Slit2-/- EBs. (D,E) GFP immunostaining on (D) Slit2-/- and (E) +/+ EBs. (F,G) GFP immunostaining on (F) Slit2-/- and (G) +/+ ducts. (H,I) Slit3-/- outgrowth stained for ß-galactosidase activity. (H) Slit3 expression in mature virgin duct. (I) Slit3 expression in aveoli during pregnancy. Arrowheads indicate examples of positively stained cells in the cap cell layer of the EB (B,D) and in the MEC layer of the duct (F,H). Arrows indicates examples of positively stained cells in the LEC compartment of the EB (D) and duct (F,H). L, lumen. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Expression patterns of Robo1 and Robo2 during mammary gland development. (A,B) ROBO1 immunostaining on (A) +/+ and (B) Robo1-/- EBs. (C,D) Robo1-/- outgrowth stained for ß-galactosidase activity in the (C) EB and (D) duct. (D) A grazing longitudinal section through a duct. Arrowheads identify examples of MECs co-expressing Robo1 (blue) and SMA (brown). (E) RT-PCR using Dutt1- and Robo1-specific primers. Lane 1, P4 cerebellum (control); lane 2, mammary gland. A 100 bp ladder (NEB) was used as a marker, as shown on the left-hand side. Dutt1 generates a 571 bp PCR fragment and Robo1 generates a 428 bp PCR fragment. (F,G) Robo2+/- glands stained for ß-galactosidase activity in the mature virgin duct (F) and aveoli during pregnancy (G). Arrowheads indicate examples of positively stained cells in the cap cell layer of the EB (A,C) and MEC layer of the duct (D,F). (G) Arrows indicate examples of positively stained Robo2-expressing cells. L, lumen. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. Loss of either Slit2 or Robo1 leads to similarly abnormal EBs. (A-G) EBs immunostained with an antibody directed against SMA to identify cap cell layers. (A,E) Wild-type (+/+) EB morphology shows tight juxtaposition of cap and LEC layers. (B-D) Longitudinal sections through Slit2-/- EBs. (B) Exaggerated space between the cap and LEC layers (double arrowhead), and dissociated cells detected in subcapsular space. (C) Loss of LECs underlying the cap cell layer (between arrows). (D) Complete disruption of EB morphology characterized by the infolding of cap cells into the LEC compartment (arrowheads) leading to lumen loss. (F,G) Longitudinal sections through Robo1-/- EBs. (F) Exaggerated space between the cap and LEC layers (double arrowheads), dissociated cells in the subscapular space and infolding of the cap cell layer into the LEC compartment (arrowheads). (G) Complete disruption of EB morphology characterized by the infolding of cap cells into the LEC compartment (arrowheads) leading to lumen loss. (A-D) Slit2-/- outgrowths were generated by transplantation with contralateral +/+ control outgrowths. (E-G) Robo1-/- and +/+ mammary glands were from littermates. L, lumen. Scale bar: 20 µm.

 

Figure 4
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  		Fig. 4. Combined loss of Slit2 and Ntn1 leads to abnormal EB morphology with characteristics of both Slit2-/-and Ntn1-/- EBs. All EBs immunostained with antibody generated against SMA. (A-C) Slit2-/-;Ntn1-/- EBs. (A) Loss of LECs underlying the cap cell layer (between arrowheads). (B) Separation of the cap cell layer from the LEC layer (double arrowhead) and breaks in the basal lamina (rectangles). (C) Complete disruption of EB morphology characterized by the infolding of cap cells into the LEC compartment (arrowhead) leading to lumen loss. (D) +/+ EB. L, lumen. Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. The basal lamina is disrupted surrounding Slit2-/-;Ntn1-/- EBs, but it is intact surrounding Slit2-/- and Robo1-/- EBs and all ducts. Immunostaining with anti-laminin 1 on EBs (A,C,E) and ducts (B,D,F). (A,B) Slit2-/- EB and duct. (C,D) Robo1-/- EB and duct. (E,F) Slit2-/-;Ntn1-/- EB and duct. Arrowheads indicate regions of intact basal lamina. Boxed region (E) indicates area where the basal lamina is disrupted. Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. Loss of both Slit2 and Ntn1 and leads to separation of epithelial cell layers in the duct. All ducts are immunostained with antibody generated against SMA. (A,B) Slit2-/-;Ntn1-/- ducts. (A) Moderate disruption characterized by sporadic loss of LECs (arrows). (B) Separation of epithelial layer (double arrowhead). (C) Table quantifying the severity of ductal disruption. Ntn1-/- ducts are normal, whereas Slit2-/- and Slit2-/-;Ntn1-/- ducts display increasingly severe disorganization. (+) Modest disruption is characterized by sporadic loss of LECs in short (~50 µm) length of duct. (++) More severe disruption is characterized by significant stretches (~50-2000 µm) of separation of myoepithelial and LEC layers. (D) Slit2+/+;Ntn1+/+ duct from contralateral transplant shows tight juxtaposition of myoepithelial and LEC layers, and an unobstructed lumen. (E,F) Loss of Slit2 (E) leads to sporadic loss of LECs (arrows) in the duct, compared with Slit2+/+ duct (F) from contralateral transplant duct. (G,H) Ntn1-/- duct (G) appears morphologically indistinguishable compared with Ntn1-/- +/+ duct (H) from contralateral transplant. L, lumen. Scale bar: 20 µm.

 

Figure 7
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  		Fig. 7. Slit2-/-;Ntn1-/- MECs are unable to form bi-layered aggregates in vitro. (A-J) Aggregates are stained with SMA (red) and DAPI (blue). Bi-layered is defined as having one or more MECs surrounding the LEC aggregate. (A) Wild-type aggregates are bi-layered, with MECs surrounding an LEC aggregate. (B) Thirty percent of Slit2-/-;Ntn1-/- aggregates are bi-layered compared with wild type. Addition of (C) 3 µg/ml or (D) 6 µg/ml of SLIT2 and NTN1 restores bi-layered structure of the aggregates. Addition of (E) 3 µg/ml, (F) 6 µg/ml or (G) 12 µg/ml SLIT2 alone partially restores bi-layered aggregate structure. Bi-layered aggregation is not restored in the presence of (H) 3 µg/ml, (I) 6 µg/ml or (J) 12 µg/ml NTN1 alone. Quantification of percentage bi-layered aggregation is below each representative aggregate picture. (K) Quantification of aggregate size in the absence or presence of SLIT2 and NTN1. Slit2-/-;Ntn1-/- cells (gray) form greater numbers of smaller aggregates compared with wild-type cells (black). In the presence of 3 µg/ml or 6 µg/ml of SLIT2 and NTN1, Slit2-/-;Ntn1-/- cells form larger aggregates (purple). They also form larger aggregates in the presence of SLIT2 alone (blue), but with NTN1 alone (orange), the aggregates remain small. Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2006