Restriction of lactase gene expression along the proximal-to-distal axis of rat small intestine occurs during postnatal development

Restriction of Lactase Gene Expression Along the Proximal-toDistal Axis of Rat Small Intestine Occurs During Postnatal Development EDMOND H. H. M. RINGS,* ANTOON F. M. MOORMAN,§ and HANS A. BuLLER*

STEPHEN D. KRASINSKI,P ERIK H. VAN BEERS,* JAN DEKKER,* ROBERT K. MONTGOMERY,? RICHARD

J. GRAND,’

*Center for Liver and Intestinal Research, Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, and §Department of Anatomy and Embryology, Academic Medical Center Amsterdam, University of Amsterdam, Amsterdam, the Netherlands; and ‘Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, New England Medical Center Hospitals, Tufts University School of Medicine, Boston, Massachusetts

Back@wund/Aims: Developmental changes of lactase activity along the proximal-to-distal axis of the small intestine are poorly understood. A study to delineate lactase gene expression at the cellular level was undertaken. Methods: The topographical regulation of lactase was studied in conjunction with sucrase-isomaltase in proximal, middle, and distal segments of O-, 7-, 14-, 16, 16, 21-, and 28dayold and adult rats using in situ hybridization, immunohistochemistry, and ribonuclease protection assays. Results: From 0 to 16 days, lactase messenger RNA (mRNA) and protein were abundant along the total length of the small intestine. However, at weaning, lactase mRNA and protein were no longer detectable in the terminal ileum. After 28 days, zones of reduced lactase expression were found in the duodenum and terminal ileum. These zones demonstrated expression of lactase protein in scattered enterocytes along the villus (patchy expression). In contrast, sucrase-isomaltase was first detected at 16 days, with patchy expression along the total small intestine; at 21 days it was abundant. Conclusions: Concordant changes in both lactase mRNA and protein detection during development suggest that the horizontal gradient of lactase enzyme expression is dependent on lactase mRNA abundance. Furthermore, zones of patchy lactase expression appear around weaning and flank the area of high lactase expression in the midintestine. Patchy expression is also found for sucraseisomaltase before weaning.

declines

around

gradient

in the small

is found

in the jejunum,

S

In adult

humans,

intestine.3

High

whereas

ileum show low or no lactase activity.

whether

or

shown that the total amount

enzyme

activity

the duodenum

and

Studies in rats have

of lactase enzyme activity

in

contrast to total protein content remains constant during postnatal development at the level found before weaning.* Although cific activity activity

the postweaning

and the regional

decrease in lactase-spe-

distribution

along the proximal-to-distal

of the specific

(horizontal)

axis of

the small intestine are well established, the mechanisms responsible for these phenomena at the molecular level are poorly Studies

understood. have

shown

that

lactase

messenger

RNA

(mRNA) levels in humans, as well as in rats, change coordinately with the amount of enzyme activity, suggesting transcriptional control of lactase expression.*-” In contrast, several studies have shown a discrepancy between lactase enzyme and mRNA levels during development in mammals, suggesting posttranscriptional trol mechanisms.73s Other studies suggest different

conphe-

notypes for the expression of lactase in the human population, which are controlled at either the transcriptional or posttranslational level.9-11 Maiuri et al.‘* have shown the presence of a patchy pattern of expression in lactase-deficient

mall intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62; subsequently referred to as lactase) plays an important role in the nutrition of newborn mammals. The enzyme hydrolyzes both lactose and phlorizin through two independent catalytic sites’ and also functions in the digestion of glycolipids in adulthood.* During development in most mammals, lactase enzymespecific activity is high around the time of birth and

weaning.

not they have elevated or low lactase levels, the enzyme activity is found in a characteristic proximal-to-distal

could be declines. renzsonn has been

adult

humans

and suggested

that

this

the mechanism by which lactase-specific activity This observation was recently confirmed by Loet a1.i3 Patchy lactase protein expression also described in rabbits and adult rats.‘*

Abbreviations used in this papec PAP, peroxidase-antiperoxidase; RNase, ribonuclease. 0 1994 by the American Gastroenterological Association OOIB-5085/94/$3.00

1224 RINGS

Around

GASTROENTEROLOGY

ET AL.

weaning,

of microvillus

a transition

membrane

that sucrase-isomaltase tivity

decreases

throughout sition

hydrolases.

activity

and

further

enables

occurs in the expression increases when lactase ac-

remains

constant

development.15

the young

rodent

solid food. The expression

It is well known at high

levels

This enzymatic to switch

tran-

from milk to

of both lactasei62’7 and sucrase-

isomaltase’8~‘9 have been described

along the cryptMItts

(vertical) axis in the small intestine. Lactase is expressed at high levels before birth. In the prenatal period, both lactase mRNA length

and protein

of the villus.”

expression

are present

A restriction

along

of 16-, 18-, 21-, and 28day-old

intestine

for the postweaning lactase protein. midpoint and eighths

region,

without

along the total length redistribution

of the villus.”

of lactase mRNA

of differentiation sentation

detectable

intestine

saline

(PBS), pH

of the enterocytes.

of lactase mRNA

base to

was detectable

This topographic

indicated

a new aspect

This postnatal

expression

7.4. Tissue

formaldehyde (40:40:20,

PBS

and

[a-3rS)deoxycytidine and [a-32P)uridine

from Du Pont-New

bosch,

the Netherlands,

was from Ilford, purchased

pre-

England.

from Sigma Chemical

Specifications Lactase

mRNA

weaning

cDNA,

investigated. specific

lactase

in conjunction

maltase.

This

localization ization,

gene

with

study along

tine during

the postnatal

and the regional

we have studied level

development

To correlate

activity

expression

postnatal

lactase

development

mRNA using

and protein

and ribonuclease

(RNase)

performed

ME) DNA

Breeding

Labo-

LMP Agaprobes

according

were to the

method.20

in situ hybridization

in rat small intestines, using

previously

we

published

The in situ hybridization procedures for lactase and sucrase-isomaltase were performed on closely adjacent sec-

reaction

Zeist, the Netherlands, Center animal facilities

of Pennsylvania,

protocols.“~*’

conditions from TN0

using a par-

(NuSieve

of the hybridization

of sections from rats of each age were processed

Animals ratories, Medical

labeling

l-

partial

rat sucrase-isomaltase

at 15°C with [a-35S]dCTP,

tions to allow comparison

Materials and Methods rats were purchased

Rockland,

To localize lactase mRNA

intes-

in situ hybrid-

rat lactase

was detected

an 827-bp

&RI-

lactase

Localization of Lactase and Sucraselsomaltase mRNA by In Situ Hybridization

of sucrase-iso-

assays.

Wistar

multiprime

at the molecular

axis of the small

immunohistochemistry,

protection

decline

of lactase

a 1.8-kb

(cDNA)

a 2.3-kb

PA). The agarose-purified

labeled overnight

of this activity,

using

a gift of Dr. P. G. Traber, University

Philadelphia,

G5

Probes

DNA

mRNA

(pRSI-1,”

rose; FMC BioProducts,

the expression

describes

along

Sucrase-isomaltase

tial rat cDNA

also has not been

gradient

the horizontal

level.

and protein

from

emulsion

(type XIV) was

Co. (St. Louis, MO).

was derected

derived

were pur-

‘s-Hertogen-

research Pronase

of Molecular

cDNA.”

axis during

water

(1350 Ci/mmol)

Nuclear,

and nuclear

London,

The exact distribution and pattern of expression of both hydrolases along the horizontal axis around the time of

the horizontal

and

(800 Ci/mmol)

England

clone,

at the cellular

acetone,

5’-triphosphate

5’-triphosphate

chased

along the vertical

mRNA

and

in 4% para-

Chemicals

Bluescript

of lactase

phosphate-buffered

methanol,

of the complementary

has not been studied

in the lumen of the

with

respectively).

PstI insert

localization

and

for in situ hybridization

axis is comparable to that of sucrase-isomaltase mRNA expression in adult human and rat small intestines.““”

The

of the proximal

was fixed by immersion

in

of the

and distal quarters

Fecal content gently

pattern

segments,

of proximal

was flushed

immunohistochemistry

mRNA

lactase mRNA

tip, whereas lactase protein

the midpoints

were examined.

small

and distal

the total

in lactase

was found only from the villus

the midvillus at the villus

of the intestine,

No. 5

rats were analyzed

and expression

The most proximal

distal halves, and the midpoints

was observed along the vertical axis after birth.

Lactase mRNA

distribution

Vol. 106,

using

aliquots

to allow comparison

ages. Hybridization

of probe

from

of mRNA

was carried

patterns.

Series

under identical

the same labeling

expression

out overnight

at different

at 44°C. The

and housed at the Academic at constant temperature and

probe concentration was approximately 0.5 ng/pL and contained 5 X 10’ cpm/pL. Slides were dipped into liquid emul-

humidity on a la-hour light-dark cycle. The rats had free access to standard chow and water. Intestinal tissue samples

sion (Ilford G5 emulsion), and autoradiography was performed after slides were stored at 4°C for 5 days. The emulsion was

were obtained

from rats 0, 7, 14, 16, 18, 21, and 28 days after

birth and from adult rats. Sections

from two animals

of each

age were studied.

Tissue Processing Total small intestines

were dissected

rapidly. Intestinal

sections were from most proximal, middle, and most distal segments of small intestine from rats 0, 7, 14, 2 1, and 28 days after birth

and from adult

rats. Nine

segments

of the small

developed at 18%.

by dipping

the slides for 4 minutes

Tissue was counterstained

into developer

and dehydrated.

were mounted in Malinol (Chroma, Stuttgart, photographed using Agfa Pan APX 25 (15 DIN) Leverkusen, Germany). Control experiments for in situ hybridization trol hybridizations with pBR322 vector DNA,

Sections

Germany) and film (Gevaert, included conwhich was la-

beled as described above. To confirm the specificity, hybridization was also performed under more stringent

in situ condi-

RESTRICTION

May 1994

tions (higher hybridization Data are presented better

preserved

and washing

temperature

for the lower stringency tissue morphology

at 5O’C).

conditions,

as previously

which

Antibodies antibody

immunohistochemistry

against

rat lactase** used for

has been described

previously.”

The

was used in the form of ascites from pristane-primed

BALB/c mice and diluted antibody

against

chemistry

in PBS. The monospecific

rat sucrase-isomaltase

has been described

polyclonal

antibody

State University,

previously.23

Shreveport,

all biosynthetic

Results

indirect

was used in

in PBS. This antibody

(PAP) technique.” fin sections. ranging

lactase antibody

the other

the sucrase-isomaltase incubations

dilutions.

antibody

without

dilution

comparable

dilution

for

guanidinium ugation

segment

lactase protein

1C).

This pattern

mRNA

isothiocyanate

through

al.** RNA was quantified A 2607 its purity integrity

as described

by optical density

was determined

bromide

somal RNA bands in nondenaturing tection

for antisense

assays were either

cially. A template from

characterized subcloned

1.8-kb

derived

rat lactase cDNA.*

Biotechnologies,

earized with E&I

scribed with T7 RNA polymerase, A mouse p-actin as a template The template

cDNA

The template

yielding

fragment (Ambion,

yielding

260 bases. The protected

200 bases. Antisense

from a previously was

was tran-

an antisense

probe

was 117 bases. Inc., Austin,

TX) served

for an antisense probe of rat p-actin mRNA. was linearized with PstI and transcribed with

SP6 RNA polymerase, mately

commer-

Inc., New Haven, CT) and lin-

before transcription.

of 186 bases; the protected

of ribo-

fragment

RNA

an antisense fragment

probes were synthesized

in 0- and

sucrase-isomaltase

tive protein

2 shows lactase mRNA expression

weaning

and immunoreac-

along the proximal-to-distal

in 14- and 28-day-old

axis

rats. At 14 days,

the proximal, middle, and distal small intestine (Figure 2A, B, and C, respectively). Similarly, at this age lactase protein is present in the same three segments (Figure

was constructed

This

intestine

of

at

BamHI site of pIBI31

into the dephosphorylated

(International

or obtained

lactase mRNA

of the small expression

was found

lactase mRNA

1.2% agarose gels.

constructed

for mature

from

Profile of Lactase Expression Around Weaning

around

and its

staining

is clearly detectable

to the tip of the villus (Figure

et

RNA probes for use in RNase pro-

BamHI fragment

a 117-bp

measurements

by A2&A2a0 ratios,

was assessed by ethidium

Templates

by Chirgwin

No

1A). from

was found at these time points (data not shown).

Figure in

buffer and purified by ultracentrif-

cesium chloride

rats.

por-

region (Figure

of lactase gene expression

the total length

7-day-old

included

was homogenized

to the midvillus

junction

antibody.

of intestine

junction

rat (Figure

can only be detected

1B). In contrast,

RNase Protection Assays A l-2-cm

as shown in the middle

the crypt-villus along

for the

results were

The optimal

was 1:2000. Controls

the primary

paraf-

in serial dilutions,

The optimal

was 1:2000, although

with

the crypt-villus

is lined by a monolayer

of a 7-day-old

At this age, lactase mRNA

peroxidase-antiperoxidase on 7-pm-thick

epithelium,

tion of the small intestine

by immunohistochemistry

were performed

1:50 to 1:3200.

In rats the small intestine

recognizes

of differentiated

Lactase was visualized

All incubations

from

obtained

was detected unconjugated

Profile of Lactase Expression in Suckling Rats

Yeh, Louisiana

forms of the protein.*s

Lactase protein the

X lo* cpm) was added to 5 /,tg of sample

polyclonal

lmmunodetection of Lactase in Rat Small Intestines using

to a proantisense

The synthesized antisense actin probe (-5.0 X lo* cpm) was added to 0.5 pg of sample RNA. Yeast tRNA was analyzed in each experiment to correct for background.

(Sucrase-isomaltase

LA.) The antibody

the form of serum and diluted

according

et a1.25 The synthesized

RNA.

used for immunohisto-

was a gift of Dr. K.-Y.

1225

5.0 X lo4 cpm/fmol;

assays were performed

by Sambrook

lactase probe (-5.0

The monoclonal

EXPRESSION

that of the actin probe was 4.0 X lo3 cpm/fmol. RNase protection

described.”

GENE

of the lactase probe was approximately

tocol described

antibody

OF RAT LACTASE

probe of approxiwas approximately using a commer-

cially available kit (Riboprobe Gemini II; Promega Biotec, Madison, WI), according to the manufacturer’s instructions. Probes were labeled using [c&~~P)IJTP. The specific activity

is present

20, E, and F, respectively).

and is expressed abundantly

In contrast,

in

Figure 2G shows

that in the proximal part of the small intestine of the 28-day-old rat, lactase mRNA is limited to the lower part of the villus. In the middle part of the small intestine, lactase mRNA is still abundant and located from the base of the villus to the midvillus region (Figure 2H), whereas no lactase mRNA is found in the distal small intestine (Figure 21). Figure 2J shows that lactase protein can no longer be detected intestine at 28 days. In contrast,

in the proximal small in the middle part of

the small intestine and similar to lactase mRNA in this segment, lactase protein is abundant (Figure 2K). Consistent with the absence of mRNA in the distal small intestine at 28 days, Figure 2L shows no detectable lactase protein. Figure 3 shows an autoradiogram of RNase protection assays for lactase mRNA expression along the proximalto-distal axis before and after weaning (14- and 2%dayold rats) in segments comparable to those in Figure 2.

1226

RINGS

GASTROENTEROLOGY

ET AL.

Vol. 106,

No. 5

Figure 1. Profile of lactase gene expression in 7-day-old rats. (A) Standard hematoxylin and azophloxine staining of intestinal sections; villi are lined by a single layer of enterocytes. (6) Expression of lactase mRNA in an area from the villus base to approximately half of the height of the villi. (C) Expression of lactase protein; immunohistochemical staining from the villus base to the tip (bar = 100 pm).

In Figure

2A, lanes 2, 3, and 4 show comparable

of lactase mRNA and distal

at 14 days in the proximal,

small intestine,

7 show lactase mRNA in Figure

respectively. decrease

intestine.

2A). As shown

in lactase

mRNA

The middle segment

is still

expresses abundant lactase mRNA, and no expression is found in the distal segment, as shown by in situ hybridization

(Figure

21). Actin

mRNA

shows little

from 14 to 28 days along the intestine

(Figure

change 2B).

Patchy Expression of Lactase and Sucrase-lsomaltase Protein in the Intestinal Epithelium sucrase-isomaltase weaning small

protein

expression

around

using

immunohistochemistry.

SA). The middle lactase expression

cytes along the vertical lactase protein segment

At

16

days, lactase protein is present in all enterocytes along the villus, as shown in Figure 4A. Patchy lactase protein

of this

segment

5B). Individual

axis of the intestine

The distal

shows entero-

express the

part of this 0.5-cm-long

shows that the lactase protein

is detectable

on

all apical membranes of enterocytes along the villi (Figure 5C). Patchy expression also was found in control experiments with different

concentrations

tern was confirmed

by staining

was uniformly

the time of

part (Figure

of the protein

on their apical surface, while others show

no lactase protein.

of patchy

at 16,2 1, and 28 days after birth in the proximal intestine,

patchy

does not show any expression

in consecutive

4 shows a detailed analysis of lactase and

Figure

this segment (Figure

middle,

Lanes 5, 6, and

at 28 days (Figure

2G, a marked

found in the proximal

levels

sections.

of antibody.

In contrast,

sucrase-isomaltase

stained in these segments

expression

The pat-

of the same enterocytes with no evidence

(data not shown).

Patterns of expression were compared for lactase and sucrase-isomaltase in adjacent sections of small intestine. In 14-day-old Figure

rats, lactase expression

2, and no sucrase-isomaltase

was as described could

in

be detected.

In 21-day-old rats, both lactase and sucrase-isomaltase mRNA were detectable in the proximal segment of the small intestine (data not shown). Both lactase and su-

expression is observed at 2 1 days (Figure 4B). Immunoreactivity is found in scattered enterocytes, but adjacent cells show no lactase protein staining. Lactase protein is

erase-isomaltase mRNA could be found in the middle part of the intestine, with a comparable pattern of expres-

no longer detectable 28 days after birth (Figure 4C). In directly adjacent sections, analysis was performed

was present

to elucidate

sucrase-isomaltase

expression.

At 16 days,

sucrase-isomaltase is characterized by patchy expression, as shown in Figure 40. At 21 days, patchiness is no longer observed in the proximal segment (Figure 4E); sucrase-isomaltase is present in all enterocytes on the villus. This pattern is maintained during subsequent development, as shown in Figure 4F at 28 days. Interestingly, in the 21-day-old rat a striking pattern of expression of lactase protein is seen. A relatively short transition zone exists in the proximal intestine, as shown in Figure 5. In a short segment of the duodenum (length, 0.5 cm), three different patterns of expression of lactase protein can be recognized. The most proximai part of

sion along the vertical axis of the small intestine; from the crypt-villus

junction

mRNA

to the midvil-

lus. Neither mRNAs were present in crypt epithelial cells. In the distal part, however, restriction of lactase expression and disappearance of the lactase mRNA signal are observed (Figure 6A). In contrast, sucrase-isomaltase mRNA remains present in the distal ileum (Figure 6B). Consequently, lactase protein is no longer detectable (Figure 6C), and sucrase-isomaltase protein is clearly detectable in an adjacent section of the same region (Figure 6D). Table 1 summarizes the immunoreactivity data obtained using monoclonal antilactase and anti-sucraseisomaltase antibodies in different segments of the small intestine around weaning. Figure 7 integrates the data

May 1994

RESTRICTION OF RAT LACTASE GENE EXPRESSION

1227

Figure 2. Lactase mRNA and immunoreactive protein expression along the proximal-ttiistal axis around weaning in 14 and 28dayold rats. (A, B and C) Lactase mRNA expression along the horizontal axis of the small intestine at 14 days. Lactase mRNA is present and expressed at high levels in the proximal (A), middle (B), and distal(C) small intestine. (D, E, F) Expression of lactase protein at 14 days. Lactase protein is present in the proximal (D), middle (E), and distal (F) small intestine. (G, H and I) Lactase mRNA expression along the ho rizontal axis of the small intestine at 28 days. In the proximal part of the small intestine, lactase mRNA can be detected only at the base of the villus (G). In the middle part of the small intestine, lactase mRNA is clearly detectable (H). No lactase mRNA is detectable in the distal small intestine (I). (J, K and L) Expression of lactase protein at 28 days. Lactase pro tein is hardly detectable along the crypt-villus axis in the proximal small intestine(J). Lactase protein is abundant in the middle part of the small intestine (K). Lactase protein is not de tectable in the distal small intestine (L) (bar = 100 urn).

shown in Figures

2-6

and Table

1 to take into account

is found

over the time course

indicated

in the terminal

the growth

of the small intestine

studied.

16 days, lactase protein is found along the

in the midintestine

of the small intestine,

both proximally

total

At

length

as indicated

dark shaded area. At 18 days a restriction in the terminal sion, as indicated

ileum,

characterized

by the light

by the

is observed

by patchy

expres-

shaded area. No protein

by white

ileum

at 21 and 28 days, as

areas. The zone of high expression is flanked

and distally,

by zones of patchiness

from the time of weaning

onward. However,

sucrase-isomaltase

protein

cannot

be de-

tected before 16 days (data not shown). Patchy expression

1228

GASTROENTEROLOGY Vol. 106, No. 5

RINGS ET AL.

is identified

along the total length

days, as indicated

(d.1Id

14

28

e--‘Qc Itn PMD

PMD

by the light

a patchy expression

% w

the proximal

of the intestine

at 16

shaded area. At 18 days

of sucrase-isomaltase

is found at both

and distal ends of the small intestine.

phenomenon

is soon replaced by expression

cytes during

the postweaning

This

on all entero-

period, as indicated

by the

dark shaded area for 21 and 28 days.

Discussion Although

186 nt around nized

the decrease in lactase-specific

the time of weaning phenomenon,

throughout

adult

total

in mammals lactase

activity

is a well-recog-

activity

remains

high

life. This decrease in specific activity,

which is obtained

by dividing

content,

reflects

several

studies

measuring

enzyme activity

possible

by protein

mechanisms.”

lactase mRNA

Several

levels have proposed

that the decrease is the result of posttranscriptional

117

trol mechanisms,‘.’

while others have suggested

conthat the

primary control is at the transcriptional level.“*5 The availability of lactase cDNA probes, as well as antibodies, now allows analysis of changes in the pattern of lactase mRNA

and protein

of expression

at the cellular

level. In

addition, correlation of the different measurements can provide an answer to the question of the level at which

260 200

the regulation

of this small intestinal

The pattern is representative length

of lactase expression of the expression

of the intestine

enzyme occurs. shown

in Figure

of lactase along

before weaning.

1 the

Lactase mRNA

and protein as well as enzyme activity are found in all segments studied in 0-, 7-, and 14-day-old rats in a similar pattern. However, as described recently in newborn rats, the expression

of lactase mRNA

the lower portion of the villus, found in all villous enterocytes.” sion along the vertical remains

constant

axis is established

throughout

is confined

to

whereas the protein is This pattern of expresafter birth and

life. The finding

suggests

either that only enterocytes at the crypt-villus junction synthesize lactase mRNA, which is degraded during mi-

1234567% Figure 3. Lactase mRNA expression along the proximal-ttiistal axis around weaning analyzed by RNase protection assays. (A) Lactase mRNA expression at 14 days in the proximal, mlddle. and distal small intestine (lanes 2. 3, and 4, respectively) and at 28 days (lanes 5, 6, and 7. respectively). At 28 days, a marked decrease in mRNA is found in proximal intestine (lane 5), the middle segment still expresses high levels of lactase mRNA (lane 6), and no expression is found in the distal segment (lane 7). (8) Actin mRNA is present in virtually equal amounts along the intestine at 14 days (lanes 2, 3, and 4) and 28 days (lanes 5, 6, and 7). Lane 1 demonstrates the unprotected lactase and actin riboprobes. Lane 8 shows hybridization of the labeled probes with tRNA.

gration

up the villus,

villus synthesize

or that all enterocytes

lactase mRNA,

along

but with different

the rates

of synthesis and/or degradation between the lower and upper halves of the villus. On the other hand, the presence of lactase protein along the total length of the villus indicates that after biosynthesis, lactase protein is inserted into the microvillus membrane as a stable enzyme. Interestingly, a comparable pattern of sucrase-isomaltase mRNA expression confined to the lower half of the villus has been described in both human and rat small intestines.1s3i9 As stated above, before weaning, lactase mRNA and protein are found along the total small intestine. However, after 14 days of life, a gradual restriction is observed

May 1994

RESTRICTION OF RAT LACTASE GENE EXPRESSION

1229

Figure 4. Immunohistochemical analysis of lactase and suerase-isomaltase protein expression around the time of weaning in the proximal small intestine. (A) At 16 days, lactase protein is present at all enterocytes along the villus. (6) Patchy lactase protein expression is observed at 21 days. (C) Lactase protein is not detectable at 28 days. (D) Patchy suerase-isomaltase expression is found at 16 days in directly adjacent sections. (E) At 21 days, sucrase-isomaltase is present in all enterocytes on the villus. (F) At 28 days, sucrase-isomaltase is observed in all enterocytes on the villus tip, although some cells at the extreme end of the villus tip are faintly stained (bar = 50 pm).

along this horizontal axis. As shown in Figure 2, expression of lactase remains high in the middle small intestine at 28 days of life, with abundant lactase mRNA and protein. In the proximal intestine at 28 days, however, hardly any lactase protein can be visualized, with some detectable

lactase mRNA

only at the base of the villi.

tative measurements of lactase mRNA, protein, and enzyme activity using RNase protection assays, rocket immunoelectrophoresis, and enzyme quantification, respectively, in combination with measurements of transcriptional rate have confirmed that the expression of lactase is predominantly

regulated

at the transcriptional

This marked decrease in lactase mRNA in this segment, compared with the proximal segment in the 14-day-old rat, is also demonstrated by the RNase protection assay

level (Krasinski et al., submitted manuscript). Restriction of lactase expression to the midintestine is accompanied by gradual reduction in the lactase ex-

in Figure 3A (lanes 2 and 5, respectively). However, the slight difference in histological presentation between

pression in the flanking regions. This gradual reduction is characterized by transient patchy protein expression, as shown in Figure 4. This pattern is similar to previously

lactase mRNA

and protein

at 28 days, which was found

only in this segment, can be attributable to technical limitations of the immunohistochemical method, or it can indicate subtle modifications of the principal mechanism of regulation. In contrast, the distal portion of the small intestine shows neither lactase protein nor mRNA. This decrease in lactase mRNA at the cellular level is confirmed by the RNase protection assays, as shown in Figure 3. Thus, primary control of lactase gene expression seems to be at the transcriptional level. Recently, quanti-

described observations in humans and animals.‘2-‘4*2” However, the initial patchy pattern of sucrase-isomaltase in the total intestine before weaning is a novel observation and suggests that patchiness may reflect a developmental regulatory phenomenon. The patchy expression was found only for lactase or sucrase-isomaltase protein and not for lactase or sucrase-isomaltase mRNA. Whether this is real or caused to technical limitations of the in situ hybridization is currently under investigation.

1230

GASTROENTEROLOGY Vol. 106, No. 5

RINGS ET AL.

Figure 5. lmmunodetection of lactase protein in the most proximal part of the small intestine of a 2ldayold rat (length of segment, 0.5 cm). Three different patterns of lactase protein expression can be recognized: (A) no lactase protein in the most proximal part of this segment; (B) patchy lactase expression in the middle part of the segment; and (C) lactase protein in all enterocytes in the distal part of the segment (bar = 100 pm).

The restriction

of lactase mRNA

from the time of weaning portant

factors.

First,

pressing

lactase during

an area comparable

onward

and protein signifies

postweaning

to that found

development

finding

amounts

of lactase mRNA

Second,

this limited

expression

This corroborates

in the midintestine

more proximally

excovers

in the total intestine

period.

out development.4

several im-

the area in the midintestine

in the preweaning of constant

found

our earlier through-

area of lactase

and patchy

could have implications

expression

for the physio-

logical conditions under which lactose is hydrolyzed, and these findings correlate well with those of others in adult humans. 12,13Around weaning, an upsurge of lactase expression occurs in a relatively short segment in the proximal intestine, of high coincides gion, activity

is seen in the midintestine,

with high levels of activity.

a decrease found

As shown stricted

as shown in Figure 5 at 21 days. A plateau

expression

in expression

in distal

in Figure

proximally

parallels

segment

which

to this re-

the decrease

in

of the small intestine.3

6, the expression and distally

Distal

of lactase

is re-

at 21 days, whereas su-

erase-isomaltase mRNA is found over the full length of the intestine. This finding indicates that in the same enterocyte

different

ble for the control development. Figure 6. Comparison between lactase and sucrase-isomaltase mRNA and protein expression in the distal small intestine of a 21day-old rat. (A) Expression of lactase mRNA in the distal small intestine; no lactase mRNA is detectable. (B) Expression of sucraseisomaltase mRNA in an adjacent section; sucraseisomaltase mRNA is present in the distal ileum. (C) Lactase protein is not detectable in the distal small intestine. (D) Sucrase-isomaltase is clearly detectable in an adjacent section (bar = 100 pm).

regulatory

factors must be responsi-

of these two disaccharidases

At later stages of development, tase mRNA and protein expression

during

the restriction in lacis even more accentu-

ated (Table 1; Figure 7). This postweaning, region-specific restriction of lactase protein at the cellular level parallels the decline of lactase mRNA abundance, indicating control of expression at the transcriptional level. Earlier studies from our laboratory also suggested this

May 1994

RESTRICTION OF RAT LACTASE GENE EXPRESSION

1. Summary of lmmunoreactivity

Table

Data for Disaccharidase

Distribution

1231

During Development

Segmentb Age (length? Lactasez2 16 days 18 days 21 days 28 days SucraseZ3 16 days 18 days 21 days 28 days

1

2

3

4

5

6

7

+ +

+ + +

+ + +

+ + +

+ + +

+ +

+/+/+ + +

(36 (48 (58 (86

cm) cm) cm) cm)

+/_

+/-

+/-

+/-

+ + + +

(36 (48 (58 (86

cm) cm) cm) cm)

+/+/+ +

+/+/+ +

+/+ + +

+/+ + +

+/+ + +

8

9

+

+

+/-

+/_

+/-

+/+/+ +

+/+/+ +

+/+/+ +

“Total length of small intestine from pylorus to coecum. “Nine segments were taken from total small intestine (see Materials and Methods). f, Continuous immunoreactivity on brush borders of all enterocytes along the vilius; +/-, staining on brush borders of random enterocytes along the villus (patchy expression); -, no immunoreactivity on brush borders of enterocytes along the villus.

primary

control

phenomenon Although

mechanism.*,’

This study

at the histological other investigators

this

also have found a decrease

in lactase mRNA

levels, specifically

around

they

weaning,

confirms

level for the first time.

suggest

in the distal

posttranscriptional

ileum and

along

the length

of the intestine.

specific factors has recently

tase and sucrase-isomaltase.2sS29 nisms also may contribute of these genes.

Other

The presence

been described

of such

for both lac-

However,

other mecha-

to the regulation

of expression

investigators

have presented

evi-

pretranslational regulation.7S27 Our data suggest that the characteristic pattern of both lactase and sucrase-isomal-

dence that the decrease in lactase-specific activity is associated with a change in the rate of enterocyte turnover.

tase expression

Changes

trol,

possibly

is more likely under by a gradient

transcriptional

in DNA-binding

confactors

in the synthesis

development

rate of lactase protein

or in degradation

of the protein,

during have also

been reported.’ The restriction be intrinsic.

the observations

21 d

S

L 20 d S Figure 7. Distribution of lactase (L) and sucrase-isomaltase (S) pm tein along the horizontal axis of small intestine in 16-, 18-, 21-, and 28-day-old rats. This graphic representation is based on data in Table 1. Numbers l-9 indicate the locations of the segments examined (see Materials and Methods), which are presented in Table 1. Disaccharidase distribution along the small intestine, as measured at the nine sites, is represented by different shadings. Changes occurring between two points are arbitrarily shown at midpoint. Bars represent intestines at relative length. The white segment represents no immunodetection. The lightly shaded segment represents patchy expression; only isolated cells express protein (as in Figure 40). The middle darkly shaded segments represent patchy expression; approximately half of the enterocytes along the villus express protein (as in Figure 48). The darkly shaded sections represent expression in all enter* cytes (as in Figure 4A). Arrow, ligament of Treitz.

recently

is not influenced

suckling, suggesting cytes and a minimal

role for luminal

concerning

imprinted programed

appears

and extended

in this study, in newborn

of lactase expression

pothesis L

of lactase gene expression

As we reported

to by

rats the pattern by prevention

of

information in enterofactors.” The hy-

positional

information

in

the intestine along the duodenal-to-colonic axis is strongly supported by data of Rubin et a1.,30 who studied fetal intestinal isografts from normal and transgenic mice for the expression absence

of luminal

of fatty acid-binding

protein

in the

factors.

This study shows the restriction of lactase gene expression along the small intestine during postnatal development around the time of weaning. Concurrently, an upsurge along ingly, zymes villus

of sucrase-isomaltase gene expression can be seen the total length of the small intestine. Interestdepending on the stage of development both enshow patchy protein expression along the cryptaxis.

References 1. Wacker H, Keller P, Falchetto R, Legler G, Semenza G. Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucraseisomaltase and with other glycosi-

1232

RINGS ET AL.

dases, the membrane anchor of lactase-phlorizin Biol Chem 1992;267:18744-18752.

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2. Bijller HA, van Wassenaer AG, Raghavan S, Montgomery RK, Sybicki MA, Grand RJ. New insights into the lactase and glycosylceramidase activities of rat microvillus membrane (MVM) lactasephlorizin hydrolase. Am J Physiol 1989;257:G616-G623. 3. Newcomer AD, McGill DB. Distribution of disaccharidase activity in the small bowel of normal and lactase-deficient subjects. Gastroenterology 1966;51:481-488. 4. Biiller HA, Kothe MJC, Goldman DA, Grubman SA, Sasak WV, Matsudaira PT, Montgomery RK, Grand RJ. Coordinate expression of lactase-phlorizin hydrolase mRNA and enzyme levels in rat small intestine during development. J Biol Chem 1990;265:6978-6983. 5. Escher JC, de Koning ND, van Engen CGJ, Arora S, Bijller HA, Montgomery RK, Grand RJ. Molecular basis of lactase levels in adult humans. J Clin Invest 1992;89:480-483. 6. Montgomery RK, Bijller HA, Rings EHHM, Dekker J, Grand RJ. Lactose intolerance and regulation of small intestinal lactase activity. In: Berdanier CD, Hargrove JL, eds. Nutrition and gene expression. Boca Raton, FL: CRC, 1993:23-53. 7. Freund JN, Duluc I, Raul F. Discrepancy between the intestinal lactase enzymatic activity and mRNA accumulation in sucklings and adults. FEBS Lett 1989; 248:39-42. 8. Sebastio G, Villa M, Sartorio R, Guzzetta V, Poggi V, Auricchio S, Mantei N, Semenza G. Control of lactase in human adult-type hypolactasia and in weaning rabbits and rats. Am J Human Genet 1989;45:489-497. 9. Lloyd M, Mevissen G, Fisher M, Olsen W, Goodspeed D, Genini M, Boll W, Semenza G, Mantei N. Regulation of intestinal lactase in adult hypolactasia. J Clin Invest 1992;89:524-529. 10. Sterchi EE, Mills PR, Fransen JAM, Hauri HP, Lentze MJ, Naim HY, Ginsel L, Bond J. Biogenesis of intestinal lactasephlorizin hydrolase in adults with lactose intolerance. Evidence for reduced biosynthesis and sloweddown maturation in enterocytes. J Clin Invest 1990;86:1329-1337. 11. Witte J, Lloyd M, Lorenzsonn V, Korsmo H, Olsen W. The biosynthetic basis of adult lactase deficiency. J Clin Invest 1990;86:1338-1342. 12. Maiuri L, Raia V, Potter J, Swallow D, Ho MW, Fiocca R, Finzi G, Comaggia M, Capella C, Quaroni A, Auricchio S. Mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia. Gastroenterology 1991;100:359-369. 13. Lorenzsonn V, Lloyd M, Olsen WA. lmmunocytochemical heterogeneity of lactasephlorizin hydrolase in adult lactase deficiency. Gastroenterology 1993; 105:51-59. 14. Maiuri L, Rossi M, Raia V, D’Auria S, Swallow D, Quaroni A, Auricchio S. Patchy expression of lactase protein in adult rabbit and rat intestine. Gastroenterology 1992; 103:1739-1746. 15. Leeper LL, Henning SJ. Development and tissue distribution of sucraseisomaltase mRNA in rats. Am J Physiol 1990;258:G52G58. 16. Rings EHHM, Biiller HA, de Boer PAJ, Grand RJ, Montgomery RK, Lamers WH, Charles R, Moorman AFM: Messenger RNA sorting in enterocytes. Colocalization with encoded proteins. FEBS Lett 1992;300:183-187. 17. Rings EHHM, de Boer PAJ, Moorman AFM, van Beers EH, Dekker J, Montgomery RK, Grand RJ, Birller HA. Lactase gene expression during early development of rat small intestine. Gastroenterology 1992;103:1154-1161.

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Received July 28, 1993. Accepted November 2, 1993. Address requests for reprints to: Edmond H. H. M. Rings, M.D., Division of Pediatric Gastroenterology and Nutrition, G8-260, Department of Pediatrics, Academic Medical Center, Melbergdreef 9,1105 AZ Amsterdam, the Netherlands. Fax: (020) 5664440. Dr. Rings is a clinical research fellow (KWO) at the Netherlands Organization for Scientific Research (NWO). This work was supported in part by Nutricia and the Irene Kinderziekenhuls Foundation, The Netherlands (H.A.B., E.H.V.B.); by National Institutes of Health (NIH) Research Grant ROl DK 32658 and by the Center for Gastroenterology Research on Absorptive and Secretory Processes (NIH grant P30 DK 34926) (R.K.M., R.J.G.); and by a NATO Collaborative Research Grant. Presented in part at the annual meeting of the American Gastroenterological Association, May 1993, Boston, Massachusetts, and pub lished in abstract form (Gastroenterology 1993;104:A643).