Multiple developmental roles of Ahnak are suggested by localization tosites of placentation and neural plate fusion in the mouse conceptus

Karen M. Downsa,*, Jacalyn McHugha, Andrew J. Coppb, Emma Shtivelmanc

aDepartment of Anatomy, University of Wisconsin–Madison Medical School, 1300 University Avenue, Madison, WI 53706 USAbNeural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK

cCancer Research Institute, University of California San Francisco, Room S333, 2340 Sutter Street, San Francisco CA 94115, USA

Received 14 May 2002; received in revised form 21 August 2002; accepted 4 September 2002

Abstract

Ahnak is a gigantic (700 kD) phosphoprotein with a unique structure whose expression and cellular localization are dynamically regulated

during cell cycle progression. Here, we report that Ahnak is localized to sites of major morphogenesis during mouse placentation and

neurulation. Ahnak was found in: (i) derivatives of trophectoderm, including chorionic ectoderm prior to and during union with the

ectoplacental cone, presumptive syncytiotrophoblast cells in the chorionic labyrinth, and giant cells at the trophoblast-uterine interface;

(ii) the allantois prior to, during, and after union with the chorion; and (iii) the tips of the neural plate during formation of the neural tube. On

the basis of these observations, we suggest that Ahnak may play heretofore unrecognized roles in tissue union during normal mouse

development. q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Ahnak; Allantois; Chorion; Ectoplacental cone; Extraembryonic mesoderm; Gastrulation; Giant cells; Invasion; Neural crest; Neural plate; Neural

tube; Neurulation; Node; Placentation; Surface ectoderm; Trophoblast stem cells; Yolk sac

1. Results and discussion

Ahnak was originally identified as an 18 kb-long messen-

ger RNA subject to transcriptional repression in neuroblas-

toma cell lines (Shtivelman and Bishop, 1992). Later,

Ahnak was recognized as desmoyokin (Hashimoto et al.,

1993), a protein enriched in the region of the desmosomal

plaques of stratified bovine epithelia (Hashimoto et al.,

1993; Hieda et al., 1989).

Changes in phosphorylation promote Ahnak’s shuttling

between the nucleus, cytoplasm and plasma membrane in

adult mammalian cell types (Nie et al., 2000; Shtivelman

and Bishop, 1993; Sussman et al., 2001). For example,

phosphorylation of Ahnak by protein kinase B leads to the

nuclear exclusion of Ahnak via a nuclear export signal

(Sussman et al., 2001) and localizes Ahnak to the inner

surface of the plasma membrane in cultured epithelial

cells that have formed mature cell–cell junctions. Similar

localization has been described for Ahnak in differentiated

keratinocytes in vivo (Masunaga et al., 1995) and in epithe-

lial cells in vitro (Nie et al., 2000). The function of Ahnak is

not known, but Ahnak’s large size and dynamic localization

patterns make it an attractive target for analysis of develop-

mental expression.

Recently, subtractive hybridization between extraem-

bryonic mesoderm and embryonic tissues suggested that

Ahnak might be preferentially expressed in extra-embryonic

mesoderm in the mouse gastrula (Kingsley et al., 2001).

Results of that study anecdotally revealed the presence of

Ahnak in allantoic mesothelium, which mediates chorio-

allantoic union (Downs, 2002; Downs and Gardner, 1995).

Because mesothelium may also promote differentiation of

the allantois (Downs et al., 1998, 2001), localization of

Ahnak to the allantois prior to, during and immediately

after union with the chorion served as a starting point for

this study. ‘KIS’ polyclonal antibodies (see Section 2)

recognized cytoplasmic and plasmalemmal, but not nuclear,

Ahnak in histological sections prepared from mouse

conceptuses between approximately 7.5 and 10.0 days post-

coitum (dpc).

1.1. Ahnak in extraembryonic tissues

The implanting mouse conceptus contains an outer layer

of trophectoderm, extraembryonic endoderm (both parietal

and visceral), and epiblast. Epiblast will form the fetus, as

well as the amnion and extraembryonic mesoderm of the

Gene Expression Patterns 2 (2002) 27–34

1567-133X/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S0925-4773(02)00349-0

www.elsevier.com/locate/modgep

* Corresponding author. Tel.: 11-608-265-5411; fax: 11-608-262-7306.

E-mail address: kdowns@facstaff.wisc.edu (K.M. Downs).

chorion, yolk sac and all of the allantois. Derivatives of

trophectoderm not only promote attachment of the concep-

tus to the uterus, but they contribute to most of the chorionic

disk of the placenta. Morphogenetic union between the

chorion and the allantois (Downs, 2002; Downs and Gard-

ner, 1995) and between the chorion and the ectoplacental

cone to form the mature chorionic disk are essential for

correct placental ontogeny in the mouse. Chorio-allantoic

union permits penetration of the fetal circulatory system

into the chorionic disk, whilst the mature chorionic disk

mediates essential exchange between the fetal and maternal

bloodstreams.

In accord with a previous report (Kingsley et al., 2001),

we found Ahnak in all mesodermal cells lining the exocoe-

lomic cavity at all stages of development analyzed, includ-

ing the amnion, the yolk sac and its blood islands, the

chorion, and the outer cells of the allantois, whether or not

the latter had epithelialized into presumptive mesothelium

K.M. Downs et al. / Gene Expression Patterns 2 (2002) 27–3428

(Fig. 1A–F). In contrast with Flk-1 and VCAM-1, which

localize with distal-to-proximal directionality to the allan-

tois during differentiation (Downs, 2002; Downs et al.,

1998; Downs and Harmann, 1997), Ahnak exhibited no

obvious polarity (Fig. 1A–C). During fusion with the chor-

ion, Ahnak persisted in the mesothelium of the allantois, and

was prevalent in its loosely organized core (Fig. 1C), with

especially high levels in basal allantoic mesoderm contin-

uous with the amnion (Fig. 1B). By 16–18-somite pairs,

Ahnak was obvious in the endothelium of the nascent allan-

toic vasculature (Fig. 1D). That Ahnak might play a role in

sites of fusion was supported by especially high levels of

Ahnak at the periphery of the chorio-allantoic fusion junc-

tion, where adhesion between allantoic and chorionic

mesothelial cells secures the umbilicus onto the chorionic

disk (Downs, 2002; Fig. 1E). Between 12- and 27-somite

pairs, presumptive syncytiotrophoblast cells found at sites

of association with Flk-1-positive allantoic endothelial cells

also appeared to stain positively for Ahnak (Fig. 1G–J, and

data not shown).

Expression of Ahnak in derivatives of trophectoderm was

observed as early as the neural plate stage (approximately

7.5 dpc). Prior to its union with the relatively negative ecto-

placental cone, chorionic ectoderm contained abundant

cytoplasmic Ahnak (Fig. 2A–C). Then, as medial chorionic

ectoderm began to fuse with the ectoplacental cone, Ahnak

became preferentially localized to the apical portion of the

chorionic cells (Fig. 2B, C). Thereafter, Ahnak was found in

the syncytiotrophoblasts of the labyrinth, as described above

(Fig. 1G-I).

Throughout all stages examined, Ahnak was also loca-

lized to the plasma membrane in many, but not all, tropho-

blast giant cells, whether or not the conceptuses had been

dissected from their implantation sites (Fig. 2E, H). In addi-

tion, high levels of Ahnak were found in the perinuclear

region and cytoplasm of the decidua capsularis which

contains large binucleate cells (Fig. 2F, I), and in the

decidua vascularis (Fig. 2F, J). Very little staining was

found in the decidua basalis (Fig. 2F, K).

1.2. Ahnak in the embryo

Ahnak’s pattern of localization was very dynamic in the

embryonic portion of the conceptus, being apparent first in

anterior mesoderm, embryonic visceral endoderm, and in

the node at the late neural plate stage (Fig. 3A). By the

headfold stage, Ahnak’s expression had spread posteriorly

in cranial mesoderm (Fig. 3B), and was found in the heart

anlage by 3-somite pairs (Fig. 3C, D, F). The primitive

streak, nascent embryonic mesoderm, and somites were

always Ahnak-negative (Fig 3C, E). Although Ahnak was

initially not detectable in the neuroectoderm (Fig. 3A–C),

by 5-somite pairs, however, it became visible at the poster-

ior surface of the trunk (Fig. 3E), in surface ectoderm and/or

neuroectoderm. By 7-somite pairs, some endothelial cells

within the head mesenchyme were positive, as was the

endoderm of the fore- (Fig. 3F) and hindgut (not shown),

and surface ectoderm overlying the neuroectoderm of the

developing hindbrain (e.g. Fig. 3F). Thus, Ahnak may be

involved in ontogeny of the heart, notochord, definitive

endoderm, surface ectoderm and/or neural tube.

By 10–11-somite pairs, the embryos under study were

undergoing axial rotation, which provided frontal views of

the prospective brain region. These revealed Ahnak in the

tips of the dorsal cranial folds prior to, during and after

closure in the putative mid-brain region between 10- and

18-somite pairs (e.g. Fig. 3G–J, and data not shown).

To determine more accurately the whereabouts of Ahnak

during formation of the central nervous system, subsequent

efforts were focused on a systematic analysis of the neural

tube during primary and secondary neurulation at the level

of the posterior neuropore (Shum and Copp, 1996). Primary

K.M. Downs et al. / Gene Expression Patterns 2 (2002) 27–34 29

Fig. 1. Ahnak in the murine allantois, components of the exocoelomic cavity, and the chorionic labyrinth. Brightfield photomicrographs localizing Ahnak alone

(panels A–E; brown color) or with Flk-1 (panels G–I; blue color) to sagittal 6 mm thick histological sections counterstained with hematoxylin (A–F) or nuclear

fast red (G–J). (A–F): Posterior, right; anterior, left. (A) Neural plate/late allantoic bud (LB) stage, approximately 7.5 dpc. Ahnak is present in the outer cells of

the allantois (al), some distal allantoic core cells, the yolk sac mesothelium (ysm), yolk sac blood islands (bi), and the mesodermal layer of the amnion (am). ac,

amniotic cavity; m, nascent allantoic mesothelium; ps, primitive streak; x, exocoelomic cavity. (B) 2-somite pair stage (approximately 8.0 dpc). Ahnak persists

in the outer cells of the allantois, some distal allantoic core cells, as well as in basal allantoic core cells continuous with amniotic mesoderm, and is visible in the

chorionic ectoderm and mesoderm (both indicated collectively by ‘ch’). (C) 7-somite pair stage (approximately 8.5 dpc) during chorio-allantoic union. Arrow

indicates where the allantois has retracted from its point of fusion with the chorion during fixation (Downs, 2002). epc, ectoplacental cone. (D) 18-somite stage

allantois (approximately 9.5 dpc) after spreading onto the chorion. Ahnak is seen clearly in a nascent blood vessel (surrounding the asterisk), other core

allantoic cells (e.g. possibly mesenchyme and/or nascent smooth muscle), and mesothelium. chl, chorionic labyrinth. (E) Chorio-allantoic fusion junction at 18-

somite pairs showing high levels of Ahnak in peripheral intermingled chorio-allantoic mesoderm (arrow) which is thought to secure the umbilicus to the

chorionic disk (Downs, 2002), as well as in the distal allantoic region (al), including allantoic endothelium, and the nascent chorionic labyrinth (chl). (F) 8-

somite pair conceptus used as a negative control with pre-immune serum. Although the allantois is not visible in this panel, it was always negative. For this and

all experiments shown in subsequent panels, additional negative controls were the omission of primary antibodies. (G–J): 27-somite pair conceptus in which

chorionic disks were snipped away from the fetus at the level of the base of the allantois. (G) Low-magnification sagittal profile of the allantois (al), chorionic

labyrinth (chl), basal zone (bz), and decidual cells (d). (H) High magnification view of Flk-1-positive allantoic vasculature surrounded by Ahnak-positive

syncytiotrophoblast cells (arrows) in the labyrinth. Some trophoblast derivatives of the basal zone exhibited Ahnak staining. mb, maternal blood. (I) Example

of a random cross section through the labyrinth showing the relationship between Flk-1-positive allantoic blood vessels and syncytiotrophoblast-stained Ahnak

(arrows). (J) Pre-immune serum negative control of a similarly staged chorionic disk. Scale bar in (J): 200 mm (A,E,I); 150 mm (B,C); 100 mm (D,H); 30 mm

(F); 50 mm (J); 20 mm (G).

neurulation involves three consecutive modes of neural tube

formation, the major difference between them being varia-

tions in bending of the neural plate (Fig. 4). Expression of

Ahnak was especially robust during Mode 1 neurulation,

being found in surface ectoderm overlying the neural

folds, in putative neural crest lying between it and the

surface ectoderm, and in the dorsal tips of the neural plate

itself (Fig. 4A, B). As the neural folds approached each

K.M. Downs et al. / Gene Expression Patterns 2 (2002) 27–3430

Fig. 2. Ahnak in derivatives of trophectoderm and the decidual swelling. Sagittal sections immunostained for Ahnak (brown color) and counterstained with

hematoxylin. Posterior, right; anterior, left. (A) Neural plate/late allantoic bud (LB) stage (approximately 7.5 dpc). Ahnak is present in both the chorionic

ectoderm and mesoderm (collectively labeled ‘ch’). Lower levels of Ahnak are present in the ectoplacental cone (epc), and in some giant cells (gc). The arrow

points to the tip of the allantois. ec, ectoplacental cavity; x, exocoelomic cavity. (B) 4-somite stage conceptus (approximately 8.25 dpc). Ahnak is present in

chorionic mesoderm (arrow) and fills the cytoplasm of peripheral chorionic ectoderm cells (ce). Arrowhead points to medial chorionic ectodermal cells where

Ahnak appears to have translocated to the apical edge of these cells, prior to chorionic fusion with the ectoplacental cone. (C) Medial section to that of (B),

showing translocation of Ahnak to the apical region of the chorionic ectoderm (arrowheads). Arrows point to Ahnak-positive chorionic mesoderm. (D) 5-

somite stage conceptus (approximately 8.25–8.5 dpc), showing fusion of Ahnak-positive chorionic ectoderm (asterisk) with the ectoplacental cone. (E)

Peripheral staining of Ahnak in secondary trophoblast giant cells (arrows) of a conceptus similar to that in (A) and dissected from its surrounding deciduum.

The arrowhead indicates the presumptive edge of a giant cell. (F–K) 4-somite pair conceptuses within their decidual swellings. (F) The arrow points to the

amnion, which separates the exocoelomic cavity (x) from the amniotic cavity of the conceptus. ‘1’ is the region of the decidua capsularis, magnified in (H,I),

showing peripheral Ahnak staining in giant cells (arrows,H) and in perinuclear regions and the cytoplasm in decidual cells (I). Arrowheads point to parietal

endoderm cells, some of which stain for Ahnak, whereas others are negative. mb, maternal blood. Asterisks in (I) indicate examples of binucleate cells. ‘2’ is

the region of the decidua vascularis, magnified in (J), showing perinuclear and cytoplasmic Ahnak, and ‘3’ is the region of the decidua basalis magnified in (K)

showing relatively low levels of Ahnak in cells of the decidua basalis. (G) Similarly-staged conceptus in its deciduum processed in the presence of pre-immune

serum alone. Scale bar in (K): 200 mm (B,C,E,H–K); 100 mm (A,D); 20 mm (F,G).

other, they became intensely stained for Ahnak (Fig. 4C, D).

Following closure of the neural tube, Ahnak was found in

surface ectoderm overlying the neural tube (Fig. 4E), as well

as within the ‘seam’ at the site of dorsal neuroepithelial

fusion (not shown, but similar to that in Fig. 3J). Similarly,

in both Modes 2 and 3, Ahnak was present in the surface

ectoderm and the dorsal tips of the neuroepithelium prior to

(Fig. 4F–I, K–M ), and during fusion of the neural folds

(Fig. 4J, N). Thereafter, Ahnak was subsequently found in

the surface ectoderm overlying the dorsal neural tube (Fig.

K.M. Downs et al. / Gene Expression Patterns 2 (2002) 27–34 31

Fig. 3. Ahnak in the gastrulating embryo and during neurulation. Sagittal (A–F; posterior, right; anterior, left) and frontal (G–J) sections stained for Ahnak

(brown color) and counterstained in hematoxylin. (A) Neural plate/late allantoic bud (LB) stage (approximately 7.5 dpc). Ahnak is localized to anterior

mesoderm (m) whose posterior limit is indicated by the arrow, embryonic visceral endoderm (arrowheads), and the node (n). (B) Conceptus at early headfold

stage (EHF, approximately 8.0 dpc) showing the relationship between Ahnak staining in the embryonic and extraembryonic regions. Patterns of expression are

similar to those described in Figs. 1 and 2, and panel 3A, but the expression domain of Ahnak in embryonic mesoderm has spread farther posteriorly (arrow).

(C) 3-somite pair stage (approximately 8.25 dpc). Ahnak is expressed in the heart anlage (asterisk), cranial mesoderm (cm), definitive (visceral) endoderm

(arrowheads) (Lawson et al., 1986; Lawson and Pedersen, 1987), but not in the somites (arrows), posterior region, or neuroectoderm (ne). (D) Head region of a

5-somite pair stage (approximately 8.25–8.5 dpc) conceptus showing Ahnak in the nascent heart (asterisk), and many cells of the cranial mesenchyme (cm). (E)

Ahnak in the putative surface ectoderm (arrow) of a 5-somite pair embryo and in the endodermal component of the trunk (arrowheads). (F) 7-somite pair

(approximately 8.5 dpc) conceptus showing Ahnak in the pericardial region (pc), cranial mesenchyme, and definitive endoderm of the foregut (asterisk). Arrow

points to staining in surface ectoderm overlying the hindbrain. (G–J) 16-somite pair cranial neural tube in the putative mid-brain region showing the presence of

Ahnak (arrows) in the dorsal tips of the neural plate before (G), during (H,I), and immediately after (J) fusion. Arrowheads indicate putative apoptotic cells.

Scale bar in (J): 200 mm (D,E,G,H,J); 150 mm (I); 100 mm (A,C,F); 50 mm (B).

4J, O) and fused seam in some sections of the nascent neural

tube (not shown).

Whilst the brain and upper spinal cord develop by

folding of the neural plate in primary neurulation, second-

ary neurulation involves cavitation of mesoderm to

produce the lower spinal cord (Copp et al., 1990). Ahnak

was not found in the neuroectoderm during secondary

neurulation (Fig. 5A–D), but anti-Ahnak antibodies did

K.M. Downs et al. / Gene Expression Patterns 2 (2002) 27–3432

Fig. 4. Ahnak in the neural plate during primary neurulation. Transversely sectioned nascent neural tubes in the region of the posterior neuropore stained for

Ahnak (brown color) and counterstained in hematoxylin. (A–E) Mode 1, 8-somite pairs (approximately 8.5–9.0 dpc). Ahnak is found at the peripheral edges of

the flattened neural plate (np) (A), which begins to bend at the median hinge point (asterisk), thereby taking on a V-shape (B), the dorsal tips of which are

Ahnak-positive. The dorsal edges approach each other (C), and begin to make contact (D) where Ahnak is present, such that in (E), fusion has occurred,

resulting in formation of a slit-shaped lumen, with Ahnak in dorsal neuroepithelium and overlying surface ectoderm. s, somites; ge, gut endoderm; n,

notochord, which is more positive at this stage than at any other. (F–J) Mode 2, 19-somite pairs (approximately 9.5 dpc). (F) Ahnak is visible at the distal

edges of the flat neural plate, which bends at both the median hinge point and the paired dorsolateral hinge points (arrowheads, G–H), leading to fusion (I–J),

and a diamond-shaped lumen. (K–O) Mode 3, 27-somite pairs (approximately 10.0 dpc). Here, the entire neural plate bends (L–M), resulting in a round lumen

upon fusion between Ahnak-positive dorsal edges of the neural plate (N–O). (P) Typical example of negative pre-immune serum control (Mode 2 neurulation).

Scale bar in (P): 150 mm (A–E); 200 mm (F–P).

bind to median dorsal cells of the closed neural tube (Fig.

5E–H).

2. Materials and methods

Polyclonal antibodies (KIS) raised against human

AHNAK peptides have been described previously (Shtivel-

man and Bishop, 1993; Sussman et al., 2001) and are reac-

tive with murine Ahnak as expected based on the identity of

the immunizing peptide sequences between human and

murine Ahnak. Mouse matings, collection and staging of

conceptuses, histology, Bouin’s based immunostaining

and isolation of neural tubes have been previously described

(Downs, 2002; Downs and Davies, 1993; Downs and Gard-

ner, 1995; Shum and Copp, 1996). All materials used here

were fixed in Bouin’s fluid for 2 h at 4 8C, followed by

subsequent rinses in phosphate-buffered saline (PBS;

Sigma) and standard methods of dehydration, clearing and

embedding in paraffin wax. All materials were sectioned at a

thickness of 6 mm. Immunostaining in Bouin’s-fixed mate-

rial was compared within the same experiments with mate-

rial fixed in 4% paraformaldehyde- and 10% formalin-fixed

material. Results revealed Ahnak only in extraembryonic

mesoderm (data not shown), as previously described

(Kingsley et al., 2001) in the latter two fixatives. For each

stage, experiments were carried out minimally in triplicate,

and included negative controls, both pre-immune serum and

omission of antibodies, with no false positive results. Care

and use of laboratory animals for this study were in accor-

dance with the University of Wisconsin Institutional Animal

Care and Use Committee and the Guide for the Care and

Use of Laboratory Animals (National Institute of Health

publication 85-23, revised 1985).

Acknowledgements

K.M.D. gratefully acknowledges funding from the

National Institutes of Health (Grants HD36847 and

HD42706), the Wellcome Trust Programme Grant 051690

to A. J. C., and the institutional support from the Cancer

Research Institute of the University of California-San Fran-

cisco to E. S.

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