Endogenous ß-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system

Abstract

Endogenous &#35-galactosidase activity has been shown in the digestive tract of amphioxus from the larval to the adult stage and it can be easily followed as a histochemical marker. Enzymatic activity first appeared in 30-h larvae, became evident in 36-h larvae and remained in adults. In situ detection of &#35-galactosidase activity was used to monitor morphological and functional differentiation of the digestive system and the posteriorization of the endodermal structures in retinoic-acid treated embryos. The endogenous &#35-galactosidase activity was distinguished from the bacterial lacZ reporter by incubation at low pH.

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Abstract Endogenous β-galactosidase activity has been
shown in the digestive tract of amphioxus from the larval
to the adult stage and it can be easily followed as a histo-
chemical marker. Enzymatic activity first appeared in
30-h larvae, became evident in 36-h larvae and remained
in adults. In situ detection of β-galactosidase activity
was used to monitor morphological and functional dif-
ferentiation of the digestive system and the posterioriza-
tion of the endodermal structures in retinoic-acid treated
embryos. The endogenous β-galactosidase activity was
distinguished from the bacterial lacZ reporter by incuba-
tion at low pH.
Keywords Amphioxus · β-Galactosidase activity ·
Endodermal marker
Introduction
β-Galactosidases (β-gal) are lysosomal enzymes that
cleave non-reducing β-D-galactose residues in β-D-ga-
lactosides. They contribute to glycolipid metabolism and
their deficiency is associated with GM1gangliosidosis, an
inherited metabolic disorder (Gossrau et al. 1991). En-
dogenous β-gal activity has been reported in Drosophila
and mammals (Lodja 1970; Schnetzer and Tyler 1996).
Although there is some intra-specific variation, tissues
that are rich in β-gal include intestine, kidney and epi-
didymis (Conchie et al. 1958; Pearson et al. 1963). Fur-
thermore, β-gal activity at pH 6.0 has been reported as a
marker for cell senescence (Dimri et al. 1995).
Results and discussion
In the research reported here, endogenous β-gal activity
has been detected in the chordate amphioxus, the closest
living relative to vertebrates. This enzymatic activity ap-
peared after staining incubation for over 48 h, at the ven-
tral posterior endoderm of Branchiostoma floridae early
larvae (30 h; Fig. 1A,B).
This was before larval feeding since the mouth had
not yet opened. At 36 h, all the larvae showed β-gal ac-
tivity in the midgut (Fig. 1C), and a strong signal was
observed from the midgut to the anus at 50 h, whereas
the pharyngeal portion remained unstained (Fig. 1D). In
addition, a conspicuous signal was detected at the mid-
hindgut junction, where the iliocolon ring develops. Al-
though the digestive tract morphologically appeared as
an uncompartmentalized tube the uneven staining pattern
of 6-day and 15-day larvae revealed functional differen-
tiation (Fig. 1E–H). The high β-gal activity at the mid-
hindgut junction was located in two circular segments
boundering the iliocolon ring (Fig. 1H). An intensely
stained region was observed in the esophagus-midgut
junction, from which the hepatic diverticulum will dif-
ferentiate after metamorphosis. Another intensely stained
small group of cells in the posterior left wall of the mid-
gut could correspond to a new landmark of asymmetric
amphioxus development (Fig. 1G,H), which could be
used to draw new body part homologies between amphi-
oxus and vertebrates (Holland and Holland 1999). The
appearance of the 11th gill slit marks the beginning of
amphioxus metamorphosis. Observations from this time
onwards showed that the β-gal pattern remained in the
gut and expanded into the developing hepatic diverticu-
lum (Fig. 1F).
The involvement of retinoic acid (RA) in chordate an-
terior-posterior axis formation is well established. Exoge-
nous RA affects endodermal development in ascidians
Edited by J. Campos-Ortega
C. Cañestro · R. Albalat · R. Gonzàlez-Duarte (✉)
Departament de Genètica, Facultat de Biologia,
Universitat de Barcelona, 645 Av. Diagonal, 08028 Barcelona,
Spain
e-mail: roser@bio.ub.es
Fax: +34-93-4110969
H. Escrivà
UMR 49 du CNRS, Ecole Normale Supérieure de Lyon,
46 Allée d'Italie, 69364 Lyon Cedex 07, France
Dev Genes Evol (2001) 211:154–156
DOI 10.1007/s004270100137
EXPRESSION NOTE
Cristian Cañestro · Ricard Albalat · Hector Escrivà
Roser Gonzàlez-Duarte
Endogenous β β-galactosidase activity in amphioxus:
a useful histochemical marker for the digestive system
Received: 8 December 2000 / Accepted: 3 January 2001 / Published online: 20 February 2001
© Springer-Verlag 2001

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(urochordates; Hinman and Degnan 1998) and induces the
loss of pharyngeal arches in vertebrates, due to Hox code
alterations in neural crest migrating cells (Kraft et al.
1994; Lee et al. 1995). In amphioxus, RA treatment af-
fects the pharyngeal development not by changing the
Hox code but by inducing the overexpression of Pax-1
and possibly other genes (Holland and Holland 1996).
This posteriorization effect could be followed by the β-gal
pattern of treated animals (Fig. 2A,B): their digestive tract
resembled a blue tube that extended along the anterior-
posterior axis (Fig. 2C,D). β-gal activity was detected in
the digestive tract but not in the pharynx of wild-type em-
bryos. Detection of β-gal activity in the presumptive pha-
ryngeal region of treated animals indicated that RA not
only prevented the formation of gill slit and mouth but
probably induced an alteration of gene expression in the
anterior endoderm and hence functional posteriorization.
The β-gal of amphioxus and mammals have acidic pH
optima. They both behave poorly at weakly alkaline con-
ditions, which are very favorable to the prokaryotic en-
zymes. On the other hand, an extensive antibiotic treat-
ment (0.5 µg/ml penicillin, 0.5 µg/ml gentamycin and
1 µg/ml streptomycin) did not alter β-gal staining. Be-
sides, β-gal activity detection preceded the mouth open-
ing. Taken together, these findings indicated that β-gal
activity is endogenous in amphioxus.
The Lac Z gene has been extensively used as a marker
in gene transfer assays. In this case, histochemical dis-
crimination of the prokaryotic activity from endogenous
β-gal activity would be attainable at pH >8.5 under the
conditions already described for mammals (Weiss et al.
1999). At these pHs the amphioxus β-gal activity was al-
ways negligible whereas Lac-Z expression of transformed
Escherichia coli DH5α was clearly identified (data not
shown). The discrimination between these two activities,
β-gal endogenous and bacterial Lac-Z, is of relevance be-
fore using this prokaryotic reporter in gene transfer assays.
Fig. 1A–I β-Galactosidase (β-gal) activity detection during Bran-
chiostoma floridae development. Animals were fixed in 0.2% glu-
taraldehyde in calcium/magnesium-free diluted artificial sea water
(308 mM NaCl, 7.3 mM KCl, 1.4 mM NaHCO3and 17 mM
Na2SO4) for 30 min at room temperature. After two washes in
PBT (phosphate-buffered saline pH 7.2, Tween-20 0.1%) for
10 min each and once in staining buffer [1 mM MgCl26H2O,
3 mM K4Fe(CN)63H2O and 3 mM K3Fe(CN)6in PBT] the sam-
ples were stained in fresh staining buffer with 0.4 mg/ml X-gal at
37°C. At alkaline pHs, 100 mM Tris-HCl buffer for the staining
solution, instead of PBT, was used. The samples were fixed in 4%
paraformaldehyde in PBT overnight at 4°C, washed three times in
PBT and mounted in 80% glycerol in PBT. All specimens were
oriented with the anterior end of the animal toward the left; in lat-
eral views dorsal is to the top. A At the 24-h larval stage, β-gal ac-
tivity was not detected. B β-Gal activity was weakly detected in
the presumptive gut of 30-h larvae. C β-Gal stained the midgut of
36-h larvae uniformly. D–F Uneven staining showed gut compart-
mentalization in 4-day (D), 6-day (E) and 15-day (F) larvae. G–H
Ventral view of 8-gill-slit larvae. Asymmetrical differentiation was
detected on the left posterior midgut. I Strong β-gal activity was
detected in the hepatic diverticulum of adult animals. a-mg Anteri-
or midgut, an anus, gs gill slits, gl gut light, hd hepatic diverticu-
lum, hg hindgut, e esophagus, eps eye pigment spot, ir iliocolon
ring, mg mid-gut, m mouth, ph pharynx, p-mg posterior midgut,
pps primary pigment spot

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Acknowledgements
Holland for helpful discussions. We thank J.M. Lawrence for labo-
ratory facilities and Ray Martinez for technical support in Tampa,
Florida (US) and Robin Rycroft for the English version. This work
was supported by grants from DGICYT to R.G. (Ministerio de
Educación y Cultura, Spain, PB96–0220, BMC2000–0536 and
UE98–0014), EU contract BIO4 CT97–2123, and a FPI fellowship
to C.C. from the CIRIT (Generalitat de Catalunya, 1997FI00007).
We are indebted to L. Holland and N.D.
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