Bmp7 Functions via a Polarity Mechanism to Promote Cloacal Septation

During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood.We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse.Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations

Potential Use of Antimicrobial Peptides as Vaginal Spermicides/Microbicides

The concurrent increases in global population and sexually transmitted infection (STI) demand a search for agents with dual spermicidal and microbicidal properties for topical vaginal application. Previous attempts to develop the surfactant spermicide, nonoxynol-9 (N-9), into a vaginal microbicide were unsuccessful largely due to its inefficiency to kill microbes. Furthermore, N-9 causes damage to the vaginal epithelium, thus accelerating microbes to enter the women’s body. For this reason, antimicrobial peptides (AMPs), naturally secreted by all forms of life as part of innate immunity, deserve evaluation for their potential spermicidal effects. To date, twelve spermicidal AMPs have been described including LL-37, magainin 2 and nisin A. Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use for the following reasons. First, it is a human AMP naturally produced in the vagina after intercourse. Second, LL-37 exerts microbicidal effects to numerous microbes including those that cause STI. Third, its cytotoxicity is selective to sperm and not to the female reproductive tract. Furthermore, the spermicidal effects of LL-37 have been demonstrated in vivo in mice. Therefore, the availability of LL-37 as a vaginal spermicide/microbicide will empower women for self-protection against unwanted pregnancies and STI

Time-line of normal cloacal partitioning in the mouse (A), and arrest in cloacal septation in <i>Bmp7</i> null embryo (C compare to B).

<p>(A) Domains of <i>Bmp7</i> expression in the urorectal mesenchyme (URM), urethral plate (up), and genital ectoderm (ge) are shown in blue. Cloacal endoderm is in green. At E10, the ventral part of the cloaca (cl) extends rostrally to give rise to the embryonic urogenital sinus and the primordium of the bladder (bl). At E12, <i>Bmp7</i> expression appears in the dorsal genital mesenchyme (gm). At E14, <i>Bmp7</i> expression in the URM shifts to the ventral portion of the genital tubercle (dashed arrow). (B, C) Histological sagittal sections of normal <i>Bmp7^lacZ/+</i> (B) and <i>Bmp7^lacZ/lacZ</i> null embryos (C) stained with X-gal. Heterozygous embryo (B) shows normal position of the rectum (r) and anus (an). In <i>Bmp7^lacZ/lacZ</i> null embryo (C), a hypoplastic rectum and urethra (ur) open into a common cloacal orifice covered by the cloacal membrane (clm). Scale bars: 100 µm.</p

<i>Bmp7</i> functions to promote cell survival in the cloacal endoderm.

<p>(A–B) Sagittal section of E12.5 wild type (A) and <i>Bmp7</i> null (B) cloacal regions were processed for TUNEL assay, and nuclei stained with DAPI. (C) Graph presents percentage ratios of TUNEL-positive cells in the cloacal endoderm (CE) and genital mesenchyme (gm). Calculations were carried out as described in Methods. Student's t-test: *P<0.01. Scale bars: 50 µm.</p

Epithelial differentiation is delayed in <i>Bmp7</i> null endoderm.

<p>Wild type and <i>Bmp7</i> null transverse cloacal sections at E11.5 (A, B) and sagittal sections at E13.5 (C, D) were labeled for Keratin 14 (K14). (A) In wild type endoderm, accumulation of K14 begins at E11.5. (B) E11.5 <i>Bmp7</i> null cloacal endoderm lacks K14. However, K14 is present in the ectoderm (ec). Arrowheads indicate the contacts between urethral plate and ectoderm. Scale bars, 100 µm.</p

Polarity of cell divisions is disrupted in <i>Bmp7</i> null the cloacal endoderm.

<p>Sagittal (A) and transverse (A′) views of murine cloacal region at E11.5. Dashed line in (A) indicates the plane of sections in (B–B″, D–D″). Dorsal, d, and ventral, v. (A′) Radial, apical-basal, direction in the cloacal cavity is defined as 90 degrees, and tangential direction, as zero degrees. Examples of transverse cloacal sections at E11.5 of wild-type (B–B″) and <i>Bmp7</i> null (D–D″) embryos labeled for pHH3 and imaged using confocal microscopy. Scale bars, 100 µm. (D, F) Images of individual wild type (D) and <i>Bmp7</i> null (F) mitotic nuclear pairs numbered in C–C″ and E–E″ shown at 63× resolution. White vectors indicate the radial direction. Red vectors in (D) and blue vectors in (F) indicate the direction of separation of the chromosome bundles determined on confocal z-sections. (G) Graphic presentation of the distributions of mitotic angles in wild type (red bars) and <i>Bmp7</i> null (blue bars) cloacal endoderm. KS test: Nwt = 172, Nmut = 168, P<0.001. (G, G′) A simple model of the axial longitudinal section of the cloaca is depicted as a circle, one cell wide and eight cells in diameter. (G) If cell divisions are oriented tangentially (double arrows), after 8 divisions the width of the epithelium remains the same and the diameter of the lumen is increased by 1.7. (G′) If cell divisions are oriented radially, after 8 divisions, daughter cells fill the section and topologically separate dorsal and ventral lumens.</p

Activity of the BMP/SMAD and JNK pathways in the human fetal urethra and rectum at 7 weeks of gestation.

<p>Transverse adjacent paraffin sections are labeled for pJUN (A) and pSMAD1/5/8 (B). Scale bars: 100 µm.</p

Loss of <i>Bmp7</i> results in a decrease in cell proliferation in the cloacal regions, and defects in epithelial stratification.

<p>(A, B) Sagittal sections of wild type (A) and <i>Bmp7</i> null (B) cloacal regions at E11.5 were immunolabed for pHH3, and nuclei stained with DAPI. Scale bars, 50 µm. Framed areas of the cloacal endoderm are shown at high magnification in the insets. Arrows indicate the width of the cloacal epithelium. (C) Graph presents the average number of pHH3+ cells in a 100 µm² square in the cloacal endoderm (ce), URM and genital mesenchyme (gm). Student's t-test: *P<0.01.</p

Activity of the Bmp/Smad and JNK pathways in the murine cloacal region.

<p>Wild-type and <i>Bmp7</i>-/- sagittal sections at E12.5 were immunolabeled for pSmad1/5/8 (A, F) and phospho-c-Jun (pJun) (B,G). Portions of sections labeled with pJun (B, G) are shown at high resolution: (C) wild type rectum, (D) wild type urethra, (G) <i>Bmp7 -/-</i> rectum, and (I) <i>Bmp7 -/-</i> urethra. Nuclei are labeled with DAPI. Scale bars: 100 µm. (E) Graphic presentation of the density of pJun+ cells in the cloacal endoderm (CE) and URM in wild-type (red bars) and mutant cloacas (blue bars). Student's t-test: *P<0.01; ** P<0.005. (J) Western analysis for phospho-JNK (pJNK) and pJun in control and <i>Bmp7</i> null cloacal tissues at E12.5. ß-actin was used as a loading control.</p