Cellular Proliferation in the Urorectal Septation

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J. Anat. (2005) 207, pp353–364

© Anatomical Society of Great Britain and Ireland 2005

BlackwellPublishing, Ltd.

Cellular proliferation in the urorectal septation complexof the human embryo at Carnegie stages 13–18: a nucleararea-based morphometric analysisJosep Nebot-Cegarra1, Pere Jordi Fàbregas1 and Inma Sánchez-Pérez

2

1

Unitat d’Anatomia i d’Embriologia, Departament de Ciències Morfològiques, and 2

Unitat de Bioestadística, Departament de

Pediatria, d’Obstetrícia i Ginecologia i de Medicina Preventiva, Facultat de Medicina, Universitat Autònoma de Barcelona (UAB),

Bellaterra, Spain

Abstract

In order to analyse the patterns of cellular proliferation both in the mesenchyme of the urorectal septum (URS)

and in the adjacent territories (posterior urogenital mesenchyme, anterior intestinal mesenchyme and cloacal folds

mesenchyme), as well as their contribution to the process of cloacal division, a computer-assisted method was used

to obtain the nuclear area of 3874 mesenchymal cells from camera lucida drawings of nuclear contours of selected

sections of human embryos [Carnegie stages (CSs) 13–18]. Based on changes in the size of the nucleus during thecellular cycle, we considered proliferating cells in each territory to be those with a nuclear area over the 75th per-

centile. The URS showed increasing cell proliferation, with proliferation patterns that coincided closely with cloacal

folds mesenchyme, and with less overall proliferation than urogenital and intestinal mesenchymes. Furthermore,

at CS 18, we observed the beginning of the rupture in the cloacal membrane; however, no fusion has been demon-

strated either between the URS and the cloacal membrane or between the cloacal folds. The results suggest that

cloacal division depends on a morphogenetic complex where the URS adjacent territories could determine septal

displacement at the time that their mesenchymes could be partially incorporated within the proliferating URS.

Key words

cellular nucleus; cloaca; computer-assisted method; development; urorectal septum.

Introduction

The mesenchymal structure that separates the lumen of

the urogenital sinus or its derivatives from the anorectum

at the cloaca is known as the urorectal septum (URS)

(Rogers et al. 2002) and is superficially coated with

respective epithelial layers. Some authors do not con-

sider it an independent structure but merely the

confluence of the urogenital sinus and hindgut mesen-

chymes (van der Putte & Neeteson, 1983; Penington &

Hutson, 2003).

Increased knowledge of the normal human embryonic

cloaca septation, aside from any advance in develop-

mental science, will enhance our understanding of the

caudal malformations associated with cloacal alterations

(Paidas et al. 1999), such as the URS malformation sequ-

ence (Escobar et al. 1987; Wheeler et al. 1997; Gripp

et al. 1999; Wheeler & Weaver, 2001; He et al. 2004). In

this way, the appearance of caudal malformations has

been related to injuries either to the hindgut endoderm

or to the primitive streak mesoderm region (Alles & Sulik,

1993; Mesrobian et al. 1994) and to alterations in the

key factors of the cloacal septation (Mills & Pergament,

1997; Kimmel et al. 2000; Roberts, 2000; Ince et al.

2002; Jo Mauch & Albertine, 2002; Dravis et al. 2004).

Classically, the cloacal septation has been interpreted

as the consequence of a crescenteric coronal fold

(Tourneaux’s fold) descent towards the cloacal mem-

brane (Tourneux, 1888), of the midline fusion of two

lateral cloacal folds (Rathke’s plicae) (Rathke, cited by

Nievelstein et al. 1998), or, in an integrating theory, as

the confluence of these three folds (Stephens, 1981;

Larsen, 2003; Dravis et al. 2004). In a more recent approach,

Correspondence

Dr Josep Nebot-Cegarra, Unitat d’Anatomia i d’Embriologia,

Departament de Ciències Morfològiques, Facultat de Medicina,

Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

E: [email protected]

Accepted for publication 5 July 2005



Cellular proliferation in the urorectal septation complex, J. Nebot-Cegarra et al.


354

the importance of URS growth has diminished and its

formation is considered to be a consequence of the

embryonic caudal folding that closes the angle of the

cloacal ceiling (Kluth et al. 1995; Nievelstein et al. 1998),

allowing the confrontation of the mesenchymal layers

of the allantois/urogenital sinus and yolk sac/hindgut

(Nievelstein et al. 1998; Paidas et al. 1999; Penington &

Hutson, 2003) and their later fusion (Nievelstein et al.

1998). Nevertheless, studies that observe the classical

point of view, explaining cloacal division as the result

of URS active growth, are still appearing (Mesrobian

et al. 1994; Kromer, 1996, 1999; Qi et al. 2000c, 2002;

Dravis et al. 2004; Hynes & Fraher, 2004). Whichever

mechanism is responsible for cloacal septation, it seems

clear that during the embryonic period there is a pro-

gressive approximation between the tip of the URS and

the cloacal membrane. This seems to be due to a com-

bination of events resulting in a transformation of the

relative position of different structures (van der Putte

& Neeteson, 1983; Kluth & Lambrecht, 1997; Nievelstein

et al. 1998; Paidas et al. 1999) and not only to the growth

of the URS (Kromer, 1996).

There is no consensus on whether the URS and cloacal

membrane fuse together. Whereas some authors have

observed this to be true in mice (Kimmel et al. 2000),

rats (Qi et al. 2000b,c, 2002; Bai et al. 2004) and humans

(Kromer, 1996; Ince et al. 2002), others have observed

that the cloacal membrane ruptures before any contact

with the URS has taken place, as in humans (van der

Putte, 1986; Nievelstein et al. 1998; Paidas et al. 1999),

mice (Sasaki et al. 2004) and pigs (van der Putte &

Neeteson, 1983). On the other hand, shortly after the

disappearance of the cloacal membrane, a secondary

occlusion of the anorectum has been described (O’Rahilly,

1978; van der Putte, 1986; O’Rahilly & Müller, 1992;

Nievelstein et al. 1998).

It seems evident that the process of cloacal septation

is still a controversial matter and new approaches are

needed to obtain more discriminative data. Because

changes in the morphology of the embryonic structures

are the result of embryonic cell dynamics (Qi et al.

2000a), cell proliferation appears to be a key factor in

determining whether the URS displays harmonic or

different growth patterns in relation to adjacent

mesenchymal territories, which could suggest passive

or active involvement in cloacal septation. Without these

data, interpretations are always excessively conditioned

by diverse factors, such as the reference points used,

the orientation of the histological section or the subtle

differences between species (Qi et al. 2000c). For these

reasons, and due to the absence of any studies that,

directly or indirectly, tackle the human cloacal septa-

tion process based on cellular proliferation, we were

encouraged to produce the present work.

To overcome the limitations of working with previ-

ously stained human embryo sections from an embryo-

logical collection, we have designed a methodology

that is able to determine the proliferating state of each

cell in accordance with the relative position of the

value of its nuclear area in relation to all the cells of its

population. We have named this methodology ‘cell

proliferation estimation based on nuclear area (CPEB-

ONA)’ and it is based on the fact that nuclear DNA and

histones are duplicated entirely in the S phase (Meshi

et al. 2000), consequently producing a progressive

increase of the nuclear size throughout the S phase

with respect to G0/G1 phase cells that has been verified

both in normal and in cancer cells (Nagl, 1977; Ormerod,

2000; Savatier et al. 2003; Miller et al. 2004). Based on

this fact it is reasonable to assert that, in a determinate

cell population the subgroup of cells with larger nuclei

includes the cells that are more likely to be in a state

of proliferation. We have carried out the study in the

interval of the embryonic period where the division of

the human cloaca takes place [Carnegie stages (CSs)

13–18] (Paidas et al. 1999). In this time interval the

mesodermal layers are still not organized, either in the

urogenital sinus and its derivates or in the anorectum

(Nievelstein et al. 1998), making the comparative study

between the various mesenchymal cellular populations

easier.

In summary, the aim of this work was to evaluate the

proliferation dynamics of different cell populations

related to cloacal septation by means of nuclear area

morphometry, in order gain a better understanding of

the involvement of each in this developmental process.

Materials and methods

Embryos

The study was carried out on a graded series of normal

human embryos belonging to the Bellaterra Collection

(Prof. Doménech Mateu). They were classified accord-

ing to O’Rahilly & Müller (1987).

The embryos were fixed in 10% buffered formalin,

embedded in paraffin and serially sectioned in the

transverse plane. Developmental data of the specimen





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as well as information on section thickness and histo-

logical staining are given in Table 1.

Descriptive conventions

In each embryo, four mesenchymal territories were

delimited: three of them in the midline of the caudal

end [preseptal, septal (URS) and postseptal] and the

fourth, constituted by a right and a left component,

in a lateral position at the cloacal walls. The borders

between the septal and the other territories were

obtained in each embryo after a meticulous analysis of

all the sections of the embryonic caudal end with the

occasional aid of linear reconstructions of the central

territories performed from lines obtained on the

sections between the centroids of the lumens of the

urogenital and digestive adjacent visceri. The centroids

were determined using Image Tool 3.0. (UTHSCSA, San

Antonio, TX, USA), available at http://ddsdx.uthscsa.edu/

dig/download.html.

We named the preseptal territory the posterior uro-

genital mesenchyme (PUM), which corresponds to the

posterior mesenchyme of the allantoic diverticulum (CS

13), the primary urogenital sinus (CSs 15–16), the sec-

ondary urogenital sinus (CS 17) and its derivatives (CS

18). The postseptal territory, named the anterior intes-

tinal mesenchyme (AIM), comprises the mesenchyme

localized in the anterior wall of the yolk-sac/hindgut

(CS 13) and the hindgut (CSs 15–18). The mesenchyme

that continues the PUM and AIM caudally, under the

celomic cavity, was considered as the septal territory

and we called it the URS based on its position separating

the urogenital and digestive lumens. The mesenchyme

of the folds in the lateral walls of the cloaca (cloacal folds)

was named the cloacal folds mesenchyme (CFM) (Fig. 1)

Morphological observation

In each embryo, the persistence of the cloacal membrane,

the presence/absence of fusion between the URS and

cloacal membrane, or between both cloacal folds and the

degree of tissue organization in mesenchymal territories

were estimated using a light microscopic approach.

Computer-assisted nuclear morphometric method

From each embryo, histological sections were selected

from a first randomly chosen section between those

that contained the URS. From this first section, the

others were selected upwards and downwards with a

sequence of one in two. If possible, a double number of

sections were selected from the PUM and AIM territo-

ries rather than from the URS. Owing to the reduced

dimensions of the cloacal folds, the number of sections

used in the CFM study oscillated between one and three

(Fig. 2).

Table 1 Data relating to the embryos and the sections studied

Developmental data of the embryos* Cross-section data

Embryo

designation CS

POA

(days)

CRL

(mm)

Thickness

(µm) Stain

SS-1 13 28 6 10 H&E

Fra-1 15 33 8, 1 8 H&EMar-2 15 33 8, 5 10 H&E

Bi-6 16 37 10 10 H&E

Pg-3 16 37 11 10 H&E

Bi-7 17 41 12 10 H&E

Re-1 18 44 15 10 H&E

*According to the catalogue of the Bellaterra Collection

(Prof. Doménech Mateu). CS, Carnegie stage; CRL, crown–rump

length; POA, post-ovulation age.

Fig. 1 Cross-section of Mar-2 embryo (CS 15), immediately

below urorectal septum (URS) (arrow marks the URS epitelial

tip). Note the outflow point of the right mesonephric duct (*)

in the urogenital sinus (UGS) and how, just over the cloaca, the

hindgut constitutes the anorectum (ar). 1, cloacal folds

mesenchyme; 2, posterior urogenital mesenchyme; 3, anterior

intestinal mesenchyme; hg, hindgut; ua, umbilical artery.

Bar = 50 µm.





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By means of the camera lucida method, using the

high-power objective (

×

100) of the Jenalumar micro-

scope (Carlss Zeis Jena, Jena, Germany) and immersion

oil, nuclear contours of 3874 mesenchymal cells were

outlined on the studied mesenchymal territories of the

selected sections. The mesothelial, vascular and blood

cells were not considered. The final magnification was

×

851.2. Each nucleus was outlined when the best focus

of its contour was reached and superimposed or bro-

ken nuclei were rejected. Camera lucida drawings were

digitalized with an HP Scanjet 3500C (Hewlett-Packard,

Palo Alto, CA, USA) and nuclear areas were determined

(

µ

m

2

) using Image Tool 3.0.

CPEBONA methology: determination of the rate of

proliferating cells

In agreement with the mesenchymal territory (four

mesenchymal territories) and the embryo (seven embryos)

to which the cells belonged, the nuclear areas were dis-

tributed in 28 groups. Because a preliminary nuclear

area analysis of the ‘no larger nucleus’ cells revealed

significant differences between territories, quartiles of

the nuclear areas were calculated separately in each

mesenchymal territory. Therefore, those cells with a

nuclear area over the Q

3

value (75th percentile) of each

territory were considered to be proliferating cells (see

Discussion). Finally, in each of the 28 groups of nuclear

areas the rate of proliferating cells was calculated as

the proportion of cells with a nuclear area over the 75th

percentile of its territory in relation to the total number

of cells of the group (cells of a territory in an embryo).

Statistics

Because of the proportional nature of the variables,

which do not have a normal distribution, comparisons

between groups were carried out using the Kruskall–

Wallis method (SPSS ver. 10.0 software, Chicago, IL, USA).

Log linear models were used to globalize distribution

of the frequencies analysed among cells of the multi-

dimensional table, generated by the cross-way of cellular

proliferation, territory and embryonic stage. A log

linear model expresses the ln of cell frequencies as an

addition of effects, each due to the categorical value of

variables that define the cell as well as the effects due

to the interactions between variables.

The model containing all possible effects is known as

a saturated model and reproduces exactly the observed

distribution of frequencies. The backward elimination

of terms produces a simple model with the restriction

that the differences between frequencies predicted

and those observed using this model must not be

statistically significant. This mechanism is not different

from the usual and well-known chi-square test of in-

dependence in a bidimensional crosstab generalized to

a multidimensional table.

Statistical significance (

P

< 0.05) of differences between

the frequencies of proliferating cells such as intrastage

and interstages were calculated based on the effects of

the log linear model referring to their variances and

covariances.

Comparative parameters of the rate of proliferating cells

Several comparative parameters were performed on

the rate of proliferating cells obtained from each of the

nuclear area groups.

Fig. 2 Diagrammatic representation of the caudal end of a CS

15 human embryo to illustrate the method used in cross-section selection (right half after sagittal section). From

among the sections in which the urorectal septum (URS) was

comprised (sections 9–12), a section was randomly chosen (e.g.

section 10). From this, the remaining sections were selected

upwards and downwards with a sequence of one in two. The

top of the sections selected above the URS was twice that of

the septal analysis (e.g. sections 2, 4, 6 and 8). Below the URS,

the sequence was applied (e.g. sections 14, 16) on the sections

which contained the cloacal folds (sections 13–16). a, allantoic

lumen; ar, anorectum lumen; c, cloaca; cf, cloacal fold;

cc, celomic cavity; cm, cloacal membrane; el, epithelial layer;

hg, hindgut lumen; ml, mesenchymal layer; ugs, urogenital

sinus lumen. Arrow: opening of the mesonephric duct.







358

Degrees of deviation of the rate of proliferating cells

The degrees of deviation of the rate of proliferating cells

per embryo were greater in the first part of the studied

interval (from CS 13 to CS 15) than in the final part,

when the rates of proliferation tended to homogeneity

between all the territories (Table 2; Fig. 5). The URS

and CFM did not show differences in the degrees of

deviation of the rate of proliferating cells per territory,

while differences were very slight between the PUM

and AIM (Table 2).

Stability index and rhythmic outlines of the rate of

proliferating cells in the territories

Along the time period studied three kinds of rhythmic

outlines of the cellular proliferation rate, related to the

stability index of the rate of proliferating cells, were

observed: the oscillating proliferation outline (PUM,

with a stability index of 17%) displayed a pattern

characterized by alternation between increases and

decreases of the cellular proliferation rate; the regular

proliferation outline (AIM, with a stability index of 50%)

where a stable rate of proliferation was predominantly

maintained; and the bimodal proliferation outline

(URS and CFM, with a stability index of 33%) that was

characterized by two phases – one initial decreasing

phase and a later increasing phase, both separated by

a stability phase (Table 2; Fig. 6).

Equality index

The greater equality index was found between the URS

and CFM (86%), with only significant differences in CS

13. Between AIM and CFM, equality began at CS 16 and

reached an index of 57%. The PUM displayed an equality

index of 29% both with AIM and with CFM, and the

URS displayed an equality index of 0 both with AIM and

with PUM (Table 3).

Fig. 3 Rate of proliferating cell (bars) and their cumulative

values (lines) of the posterior urogenital mesenchyme (PUM),

urorectal septum (URS), anterior intestinal mesenchyme (AIM)

and cloacal folds mesenchyme (CFM). Note that the

cumulative cellular proliferation at the supraseptal territories(PUM and AIM) is always higher than at the septal level.

CRL, crown rump length; CS, Carnegie stage.

Fig. 4 Comparison between rates of proliferating cell in the

urorectal septum (URS) and in cloacal folds mesenchyme

(CFM). Note the strong parallelism between both outlines.

CRL, crown rump length; CS, Carnegie stage.

Fig. 5 Degrees of deviation of the rate of proliferating cell

in all territories per embryo. CRL, crown rump length;

CS, Carnegie stage.





359

Discussion

During prenatal human development, cavities of dif-

ferent organs are divided by means of septation pro-

cesses. Septum formation could be either due to the

growth of the parietal components into a lumen or cavity

(e.g. conotruncal and auriculoventricular septation,

growth of nasal septum or palate formation) or to the

progressive development between cavities of an inter-

position tissue produced by cavity expansion (e.g. pars

muscular of interventricular septum) (Sadler, 2004). In

the tracheo-oesophageal septum, the interposed tissue

is the consequence of the formation and caudal growth

of the respiratory primordium in front of the foregut

(Zaw-Tun, 1982), with an initial separation and a later

approximation between them (Nebot-Cegarra et al.

2001).

The human cloaca also undergoes a division as a con-

sequence of URS formation. The nature of the human

cloacal septation process has been explained in diverse

and conflicting ways. On the one hand, Kromer (1996)

considers that it is the active descent of the URS that

completes the cloacal septation, whereas others have

interpreted the participation of the URS in the septa-

tion process as passive (Nievelstein et al. 1998; Paidas

et al. 1999; Rogers et al. 2002) conditioned by changes

in the position of caudal structures. Participation in the

process of the cloacal folds has been accepted [Stephens,

1981; Kromer, 1996; Rathke (cited by Nievelstein et al.

1998); Kromer, 1999; Larsen, 2003] except by Rogers

et al. (2002).

As can be inferred from the previous paragraphs in

this discussion, and from the introduction, despite

several descriptive studies, the cloacal septation is still an

open and disputed subject. We consider that determin-

ing whether participation of the URS in the cloacal sep-

tation is due to its active growth or to passive changes

of position must be answered by analysing the cellular

proliferation in the URS in relation to its neighbouring

mesenchymal territories, in order to determine if the URS

has a distinct growth pattern. The cellular proliferation

in the URS has been studied in rat (Qi et al. 2000a,c) and

mice embryos (Kubota et al. 1998; Hynes & Fraher, 2004)

with conflicting results, but never in human embryos.

This lack of information is probably related to the

fact that the majority of human embryos available for

study belong to collections of stained histological

sections that allow for the visualization of microscopic

characteristics of the tissue such as metaphasic cells

or apoptotic bodies (Nishikori et al. 1999; Matsumoto

et al. 2002), but make them unsuitable for carrying out

Table 3 Equality index (EIRPC) between the rate of proliferating cell of two territories

Compared

territories

Significant difference (P < 0.05) between the rate of proliferating cells of the compared territories

CS/CRL (mm)

13/6 15/8.1 15/8.5 16/10 16/11 17/12 18/15 EIRPC (%)

PUM/URS NO YES NO YES YES YES NO 0PUM/AIM NO NO YES NO YES NO YES 29

PUM/CFM YES YES NO NO YES NO YES 29

URS/AIM NO YES YES NO YES NO YES 0

URS/CFM YES NO NO NO NO NO NO 86

AIM/CFM NO YES YES NO NO NO NO 57

AIM, anterior intestinal mesenchyme; CRL, crown–rump length; CS, Carnegie stage; CFM, cloacal folds mesenchyme; PUM, posterior

urogenital mesenchyme; URS, urorectal septum.

Fig. 6 Simplified curves (values with no significant differences

have been considered equal) of the rate of proliferating cell.

They represent the rhythmic proliferating outlines of the

territories, from Carnegie stage 13 (left end) to Carnegie stage

18 (right end). AIM, anterior intestinal mesenchyme; CFM,cloacal folds mesenchyme; PUM; posterior urogenital

mesenchyme; URS; urorectal septum.





360

cytometric flow techniques, using bromodeoxyuridine

(BrdUrd), PCNA (Morris & Mathews, 1989), Ki-67 (Key

et al. 1993) or terminal deoxynucleotidyl transferase

biotin-dUTP nick end labelling (TUNEL) (Gavrieli et al.

1992) that could potentially provide more concrete

information about the proportion of cells that are in

the different cell cycle phases and also about the fate

of the cells. The CPEBONA methodology is a useful tool

for solving this problem because it can estimate pro-

liferation activity in a cellular population by means of a

morphometric analysis of nuclei obtained from digital-

ized conventional histological sections.

We applied the CPEBONA methodology in two steps:

first we grouped the nuclear areas of all the cells in

each territory and those whose nuclear areas were over

the 75th percentile of all areas of the territory, and

considered these to be proliferating cells. In a second

step we applied the value of the 75th percentile for the

whole territory, obtained in the previous step, to each

of the embryos involving this territory to obtain the

number of proliferating cells for each group (territory

per embryo). The ratio of the number of proliferating

cells in each group and the total number of cells for the

group is defined as the ‘rate of proliferating cells’,

which depends on the proliferation activity.

Use of the CPEBONA methodology is based on two

premises: first there is an increase in the nuclear size

during the S phase, due to duplication of DNA and his-

tones inside the nucleus, such that the average nuclear

area in the G2/M phases becomes approximately

1.5 times greater than in G0/G1 (Ormerod, 2000; Savatier

et al. 2003). The range of intermediate sizes between

G0/G1 and G2/M corresponds to those cells that are in

the S phase having different grades of DNA and histone

duplication. The second premise is that in a homogene-

ous and non-synchronic population, cells are distributed

in the different cell cycle phases in a proportion that

is directly related to the duration of each phase. For

a ‘typical quick human cell’, such as non-synchronous

embryonic cells (Murray & Hunt, 1993), with a cycle

duration of 24 h, the lengths of the different phases

are: G1, 11 h; S, 8 h; G2, 4 h; M, 1 h (Cooper & Hausman,

2004). If we consider that cells are distributed uni-

formly throughout the cycle, cell distribution should be

directly related to the length of each phase: 46% in G1,

33% in S, 17% in G2 and 4% in M. Combining the two

premises we can assume that 25% of cells with a larger

nuclear area have a high probability of being in the more

advanced cell cycle phases (final part of S, G2 and M).

Some considerations must be borne in mind in rela-

tion to the theoretical basis of the CPEBONA method-

ology. First, the length of the different cell cycle phases

changes depending on the type of organism and also

on the cell type in a particular organism (Murray &

Hunt, 1993). Secondly, not all the cells in a population

cycle at a particular moment, meaning that the pro-

liferation rate is the proportion of cycling (G1, S, G2 and

M phase) and resting cells (G0 phase) (Pardee, 1989),

which depends on the tissue type and on other factors

such as life stage, metabolic conditions or the existence

of pathological conditions (Pardee, 1989). Both in mor-

phometric determinations and in flow cytometry, G0/

G1 cells are grouped together because they display the

same nuclear size and DNA content. Moreover, G0/G1

cannot be very different because cells can switch from

one to the other depending on cellular and extracellular

factors. In this way, in CPEBONA, resting cells are inter-

mingled with G1 cells, moving the influence of non-

cycling cells to the small cell side and consequently

reinforcing the importance of the larger cells. The group

of transition cells from small G0/G1 cells to large G2/M

cells, S phase cells, comprises progressively enlarging

cells that can be divided into an initial component that

is not distinguishable from G0/G1 and an advanced

component of similar dimensions to G2/M cells; in its

proliferating group CPEBONA selects the latter because

it is possible that there is an overlap between G0/G1

cells and the initial S phase cells. With regard to the

group of M phase cells, we could only include in our

study those which are in the prophase, having an intact

nuclear envelope; this was confirmed by means of the

camera lucida method.

We can assume that despite different cell populations

having different cell distribution patterns throughout

the cell cycle, placing the division line at the 75th per-

centile ensured that no G0/G1 cells would be included

in the proliferating group (Fig. 7). On the other hand,

if the cell dynamics of a specific population is well

known, it could be possible to adjust the borderline

between ‘proliferating’ and ‘non-proliferating cells’

to provide a better adaptation of the method to the

studied population.

In addition, not all cycling cells reach the end of the

cycle and complete their division because some of are

stopped in cycle checkpoints (Hartwell & Weinert, 1989)

in G1, at the end of G2 and in the middle of the M phase,

thereby modifying their cellular fate by avoiding division

of damaged cells. Methods that consider as proliferating





361

cells only those that have reached the S phase, like CPE-

BONA, avoid including cells that stop their cycling at the

G1 checkpoint, and only those that are stopped in G2

and M checkpoints are incorrectly considered as pro-

liferating cells.

Not all cellular proliferation methodologies indicate

the same cell cycle phase, thus Ki-67 labels all cells that

are not in G0 phase (Key et al. 1993); PCNA increases in

cycling cells to reach a maximum in the S phase (Morris

& Mathews, 1989); BrdUrd, administrated exogenously,

is incorporated into the S phase nuclei (Begg et al. 1985);

and our methodology, CPEBONA, is able to recognize

cells in the second half of the S, G2 and initial M phase,

before the nuclear envelope breaks up and chromo-

somes become individualized.

In conclusion, CPEBONA is a reliable method for

determining the degree of cellular proliferation in tissue

because, although it does not select all proliferating

cells, it selects those that have the highest probability

of being in a state of proliferation. This underestima-

tion does not reduce the ability of the methodology to

compare cellular proliferation among different popu-

lations because they are all analysed by means of the

same strategy.

We have carried out the study in an interval between

CS 13 and CS 18, because this includes the seven first

post-fertilization weeks when cloacal septation occurs

in humans (Nievelstein et al. 1998). Also in this period,

mesodermal cells in the studied territories are in an

undifferentiated mesenchymal state constituting poorly

organized tissues, which make the study easier than in

more complex tissues. It is not until CS 18, the end-point

of our study, that some promyoblasts and myoblasts

can be observed, scattered in the mesoderm around

the rectum (Nievelstein et al. 1998), and it is not until

8–12 weeks after fertilization when definitive differ-

entiation in tissues and organs appears (Nievelstein

et al. 1998). The boundary between these two periods

is defined by the rupture of the cloacal membrane

(Nievelstein et al. 1998).

The URS is formed at CS 12/13 at the meeting point

of the allantoic diverticulum and yolk sac (Kromer,

1996, 1999; Nievelstein et al. 1998; Paidas et al. 1999;

Rogers et al. 2002), and as observed here. The URS origin-

ates from the allantois and yolk sac extra-embryonic

mesoderm (PUM and AIM, respectively), which, because

of the embryonic sagittal folding, are incorporated inside

the embryo (O’Rahilly, 1978; Nievelstein et al. 1998; Paidas

et al. 1999; Sadler, 2004). In this way, both components

form an angle, facing each other, and subsequently

fuse together closing the most caudal part of the celomic

cavity (Nievelstein et al. 1998). We have observed the

cloacal lateral folds (Rathke’s plicae) earlier (CS 13)

than Kromer (1996) (CS 14) but, applying the criteria of

Penington & Hutson (2003), fusion among them in all

the studied embryos has been discarded. In accordance

with other authors (van der Putte & Neeteson, 1983;

Penington & Hutson, 2003), we have observed that the

URS is continued above by the PUM in front and the

AIM behind, and below, in the walls of the undivided

cloaca, by the CFM. Progressively, the URS divides the

cloaca into a ventral part, the primary urogenital sinus,

and a dorsal part, the anorectum (CS 15) (O’Rahilly &

Müller, 1992). The mesonephric duct orifices in the

primary urogenital sinus are the boundary points that

divide this cavity into two parts: cranially, the vesico-

urethral canal (future bladder and intramural urethra)

and caudally, the definitive urogenital sinus (CS 17)

(O’Rahilly & Müller, 1992; Kromer, 1996). The definitive

urogenital sinus is in turn divided into two parts (CS 18):

the pelvic portion (future prostatic and membranous

urethra in males, and urethra and vagina in females)

and the phallic portion, including urethral plate (future

spongy urethra in males, and vestibule and labia minora

in females) (O’Rahilly & Müller, 1992; Kromer, 1996).

Fig. 7 Distribution of cell cycle phases in several embryonic

cell populations: brain (b), face (f), limbs (l) and heart (h) from

chick embryos (CH) (Heringová et al. 1998); dorsal half (d) or

ventral half (v) from the tail bud of 21–25 somites (1) or 26–30

somites (2), mouse embryos pertaining to diverse mousestrains [curly tail (CT), recomb-2 (R), BALB/c (B) and CBA/J (C)]

(Peeters et al. 1998). The 75th percentile (Q3) level is shown by

the horizontal line.





362

Although an approach between the URS and the

cloacal membrane has been observed before the rupture

of the cloacal membrane, we, like others (Nievelstein

et al. 1998; Paidas et al. 1999; Rogers et al. 2002; Sadler,

2004), have not observed fusion between them.

We observed that, throughout the embryonic inter-

val studied here, the cellular proliferation rate tends

towards homogeneity between all territories studied

(Table 2; Fig. 5) and the URS displays a tendency to

increase its proliferation rate to reach similar values to

the other mesenchymal territories in the final part of

the interval studied (Table 2; Figs 3 and 4). Together

with the fact that the URS shows progressive enlarge-

ment during development and that it takes part in the

formation of digestive and urogenital subperitoneal

visceral walls and of the rectovesical septum in men

and rectovaginal septum in women, these data suggest

that the definitive URS comes from the incorporation

of mesenchymal tissue from the adjacent territories

(PUM, AIM and CFM) into the primitive URS mesen-

chyme. It is possible that the trend towards homogeni-

zation in the proliferation rates between territories

(Fig. 5) and the progressive increase in the URS (Table 2;

Fig. 3) could be due to a mixture of process between

primitive URS cells and cells from extraseptal territories.

Migration processes of mesenchymal cells towards the

URS have been described in mice embryos (Dravis et al.

2004). It is credible to think that growth of the urogenital

viscera and hindgut tends to bring them near to each

other, just over the primitive URS, favouring contact

and subsequent fusion of opposite mesenchymes. In

this way the initial union between the allantois and

yolk sac (Nievelstein et al. 1998) could be extended,

further closing the Douglas pouch, and extending up

the URS. In the lower part of the septation complex,

the URS, located above, and cloacal folds prolonging it

downwards on both sides delimit a space or isthmus

that is being progressively closed, probably due to

the inclusion of the cloacal folds in the URS while it

descends. In this way, the URS is enlarged caudally, and

the cloacal folds could act as ‘rails’ that guide the

closure of this part of the septum. It must be emphasized

that the URS and the CFM have demostrated almost

identical proliferation behaviour over most of the stud-

ied period (Table 3; Figs 4 and 6) and it seems reason-

able to believe that they are developed together early

as components of the same structure (Kromer, 1996; Qi

et al. 2000c, 2002) and/or are under the control of the

same regulatory factors. On the other hand, the link

between URS and supraseptal territories (PUM and AIM)

makes it credible to attribute the septal descent not

only to its intrinsic growth but mainly to preponderant

global proliferation of supraseptal mesenchymal cells

(Fig. 3), as has similarly been explained for rat embryos

(Penington & Hutson, 2003). For all these reasons our

proposal is to name all of the definitive sources of URS

as the ‘urorectal septation complex’, because they

constitute a morphogenetic unity in their cellular origin

as well as in their mutual influence on developmental

changes.

In the majority of sagittal section images (van der

Putte & Neeteson, 1983; Nievelstein et al. 1998; Kimmel

et al. 2000; Qi et al. 2000a,b,c, 2002; Penington &

Hutson, 2003; Bai et al. 2004), the URS tip appears slightly

faced to the front, which could be due to displacement

in this direction as a consequence of the slight predom-

inance of cellular proliferation of the AIM over that of

the PUM, as observed in our study, especially from CS

16 onwards (Table 2; Fig. 3). These data and others

(Alles & Sulik, 1993) might indicate that an abnormal

change in the proliferation and/or apoptotic degrees in

these mesenchymal populations could modify the nor-

mal descent of the septum or the orientation of its

tip; it would therefore be interesting to corroborate

this hypothesis in embryos with septal malformations

(Nebot-Cegarra & Domenech-Mateu, 1989). Against

this hypothesis is the fact that in mouse embryos with

cloacal malformations, disturbances in the cellular

dynamics were observed in the cloacal membrane and

dorsocaudal region but not in the URS and its adjacent

territories (Kubota et al. 1998).

In conclusion, our study has enabled us to determine

that the cloacal membrane begins its rupture at CS 18

and that there is no fusion either between the URS and

cloacal membrane or between the cloacal folds them-

selves during the cloacal septation period (CSs 13–18).

Furthermore, the data regarding cellular proliferation

of the URS and its adjacent mesenchymal territories

that we have obtained allow us to consider that the

division of the human cloaca is a consequence of a

coordinated process of septation, in which the compo-

sition of the URS could be modified not only by its

intrinsic cellular proliferation but also by the incorpo-

ration into the septum of cells coming from the neigh-

bouring territories, and its position could be changed

by the differential growth of these cellular populations.

We therefore suggest that the URS and its adjacent ter-

ritories constitute a septation morphogenetic complex.





363

Acknowledgements

We thank Prof. Josep M. Domènech-Mateu (Unitat

d’Anatomia i Embriologia, UAB) for his advice in the

selection of human embryos, and Prof. Miguel Martin-

Mateo (Unitat de Bioestadística, UAB) for assistance

with the statistics, as well as Mr David Carrera for his

technical assistance. We are also grateful to Servei de

Traduccions i Revisions de Textos (UAB) for assistance

with the translation.

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Cellular Proliferation in the Urorectal Septation