/. Embryol. exp. Morph. Vol. 34, 2, pp. 373-386, 1975
Printed in Great Britain
373
Activities of haem synthetic enzymes in blood cells
of pre-natal flexed-tailed (///) anaemic mice
By R. J. COLE, 1 J. GARLICK 1 AND E. M. CHEEK 1
From the Genetics and Development Group,
School of Biological Sciences,
University of Sussex
SUMMARY
Levels of activity of 5-aminolaevulinate synthetase, 5-aminolaevulinate dehydratase, porphyrinogen synthetase and haem synthetase in circulating reticulocytes of pre-natal FL4/Re
+ / + and congenitally anaemic FLl/Re///mice have been determined. The activities of
5-aminolaevulinate synthetase and 5-aminolaevulinate dehydratase were found to be decreased in proportion to the hypochromia observed in mature liver-derived erythrocytes in
neonatal///mice, but activities of enzymes later in the haem synthetic pathway were relatively
undisturbed. No significant differences were found in levels of haem synthetic enzymes in
foetal livers of + / + and ///mice. These results indicate that the severe anaemia of late prenatal and neonatal / / / mice is due both to reduction in haem synthesis expressed at the
reticulocyte stage of erythroid differentiation and to restricted production of erythroid
progenitor cells. Retarded foetal growth and skeletal abnormalities, both characteristic of
the pre-natal expression of the///gene complement, can also be related to reduced levels of
haem synthesis, but the abnormal distribution of pigment cells seen in///animals appears
to be a secondary effect of reduced tissue oxygenation resulting from pre-natal anaemia.
INTRODUCTION
Mutation at the 'flexed tailed'' locus in mice causes severe perturbation of
pre-natal erythropoiesis with effects expressed at several levels within the developing erythroid system. The livers of early homozygous 'flexed' foetuses contain
fewer pluripotent haemopoietic stem cells than normal (Bateman & Cole, 1972)
and the development of the foetal liver as an erythropoietic organ is retarded,
suggesting restricted flow of///erythropoietic cells through the 'progenitor'
cell compartment. Studies on erythropoiesis of///foetuses in vitro (Bateman,
Cole, Regan & Tarbutt, 1972) and in vivo (Tarbutt & Cole, 1972) show that
enhanced proliferation of their liver erythroblasts occurs, although the absolute
number of blood cells produced is still less than normal. These observations
suggest that the///foetal erythropoietic system can 'recognize' and respond to
deficiencies in blood cell or haemoglobin production, and that the proliferative
1
Authors' address: Genetics and Development Group, School of Biological Sciences
University of Sussex, Falmer, Brighton, U.K.
374
R. J. COLE, J. GARLICK AND E. M. CHEEK
activity of///erythroblasts is basically normal although that of earlier precursors
may be restricted. Estimates of relative rates of iron incorporation into haem in
nucleated erythroid cells from foetal livers showed little difference in the ability
of newly explanted / / / and normal cells to utilize exogenous iron for haem
synthesis (Bateman et al. 1972). However, since a significant proportion (•£-§)
of the final haemoglobin content of mature liver-derived erythrocytes is synthesized after loss of the erythroblast nucleus, abnormalities expressed mainly
during the reticulocyte stage of erythropoiesis could have severe effects on the
overall haematological state of///foetuses.
In contrast to the apparent normality of their liver erythroblasts, circulating
erythroid cells produced by the / / / foetal liver are very abnormal. Mature
erythrocytes are reduced in size, contain a reduced complement of haemoglobin
and are characterized by inclusions of non-haem iron, indicating derangement
of haemoglobin synthesis (Gruneberg, 1942). Studies of///pre-natal reticulocytes in vitro have shown that, while iron uptake is normal, utilization of
intracellular iron for haem synthesis by intact cells is halved, and porphyrin
synthesis is also reduced (Cole, Regan & Tarbutt, 1972). Restricted production
of haem leads to derangement of the usually co-ordinated a and /? globin chain
synthesis, and / / / reticulocytes are characterized by a deficiency in /?-globin
chain synthesis with relative over-production of a chains (Cole, Garlick &
Tarbutt, 1974) Previous studies have indicated that reticulocytes of FLl/Re
fIf mice have normal levels of haem synthetase activity, but appear to be deficient
in protoporphyrin, an immediate precursor of haem, suggesting that early steps
in the haem synthetic pathway may be aberrant (Kreimer-Birnbaum et al.
1972; Cole et al. 1974). Data presented in the present paper indicate that activities of both #-aminolaevulinate synthetase and #-aminolaevulinate dehydratase
are reduced in / / / reticulocytes, and that these lesions could contribute to the
severe anaemia of pre-natal///mice.
MATERIALS AND METHODS
Animals. FL/1 Re///£v 6 /Lv 6 and FL/4 Re + / + Lvb/Lvb mice were obtained
from the Jackson Laboratory, Bar Harbor, U.S.A. The Lv locus determines
the level of #-aminolaevulinate dehydratase in a variety of pre- and post-natal
tissues. Adults of'Edinburgh'///and + / + stocks used in earlier kinetic studies
have liver levels of 8ALA dehydratase similar to FL/1 Ref/fLvblLvb and FL/4
Re + / + Lvb/Lvb and are therefore comparable to the Lvb/Lfvb genotype (FL/1
and FL/4 Re + / + LvbILvb, 2 jnmo\ PBG produced/g liver/h;
ReflfLvbILvb
Edinburgh///2-6 y^mol, + / + 1-3/flnol. Foetal material was obtained from
natural matings; the morning on which mating plugs were found defines day 0.
Tissues. Circulating foetal blood cells were harvested in normal saline, washed,
and when necessary stored under nitrogen at - 40 °C. Reticulocytosis was
measured after staining with brilliant cresyl blue, and RNA content determined
Haem synthesis in f/f mice
375
by a modified Schmidt-Tannhauser procedure. Foetal livers were dissected into
chilled normal saline.
8-Aminolaevulinate synthetase. Activity was estimated by the methods of
Freshney & Paul (1970). Extracts of foetal liver and reticulocytes were prepared
by hypotonic lysis in an equal volume of 001 M phosphate buffer, pH 7-0,
containing 20 mM-MgCl2, 10 mM EDTA, 1 mM glycine and 4 mM mercaptoethanol. Sixty fi\ samples of lysate were incubated with equal volumes of 0-04 M
phosphate buffer, pH 7-0, containing 1 mM glycine including 12/^Ci/ml [2-14C]glycine (51 mCi/mmol.), 0-27 mM pyridoxal phosphate, 3-3 mM sodium malate,.
and 1-3 mM-MgCl2. After 1 h incubation, labelled aminolaevulinate was isolated
by thin-layer electrophoresis on' Chromar 1000' glass-fibre/silica-gel mat, eluted,.
and radioactivity determined.
8-Aminolaevulinate dehydratase. Activity in reticulocytes and liver was measured by spectrophotometric determination of porphobilinogen produced from
aminolaevulinate in homogenates in phosphate buffer at pH 6-8 in anaerobic
conditions using Ehrlich's reagent (Battistino et al., 1971). Loss of porphobilinogen was measured in similar homogenates supplied with 2 X 1 0 ~ 4 M
porphobilinogen and that remaining after incubation determined spectrophotometrically as above.
Porphyrinogen synthetase. Utilization of porphobilinogen by reticulocytes
was determined in homogenates in phosphate buffer, pH 7-65, supplied with
2x 10~ 4 M porphobilinogen (Levin & Coleman, 1967) using Ehrlich's reagent.
Haem synthetase. Activity was estimated by the method of Freshney & Paul
(1971). Tissue extracts were prepared by homogenization in 1 ml 0-15M-KC1
containing 0-4 (v/v) Tween 20, and/or by freezing and thawing; 0-1 ml of
extract was incubated with 0-2 ml 0-1 M glutathione in 0-23 M Tris/HCl buffer,
pH 7-4, for 20 min; 0-05 ^Ci of [59Fe]Cl3 was added in 0-2 ml reaction mixture
and incubation continued for 1 h under nitrogen. Haem was extracted into acid
ethyl methyl ketone and radioactivity determined.
RESULTS
8-Aminolaevulinate synthetase activity in pre-natal reticulocytes
Levels of (^-aminolaevulinate synthetase activity found in circulating blood
cells of 16 to 18-day FL/4 Re + / + and FL/1 Re///*pre-natal mice are shown
in Table 1. Significant levels of activity were found only on day 16, when all the
circulating red cells contain large amounts of RNA, and it appears that formation of aminolaevulinate is very low in the circulating red cells of later foetuses.
#-Aminolaevulinate formation in reticulocytes of 16-day / / / foetuses was
reduced to 53 % of that seen in wild-type cells on a per cell basis and, although
less severe, there was also a marked deficiency of synthesis in / / / cells when
expressed per unit of RNA. ( + / + 221 ±20 and///117 ±23 nmol ALA formed/
1010 cells/h; + / + 0-36 and///0-24 nmol/ALA formed/ODU RNA/h.)
24
E M B 34
16-day reticulocytes
Expt. 1
Expt. 2
Expt. 3
Expt. 4
Expt. 5
Means
17-day reticulocytes
Expt. 1
Expt. 2
18-day reticulocytes
Expt. 1
Tissue
005
002
5-0
8
0-46
0-43
0-35
0-33
0-23
0-36
14
14
117±23
176
113
95
72
ALA formed ALA formed
(nM/ODU/h) (nM/1010 cells/h)
40
60
6-5
60
60
60
—
RNA
ODU/10 3
20
279
279
212
198
140
221 ± 20
ALA formed
(nM/1010 cells/h)
A
FL/4Re +/ +
001
40
0-5
0-7
0-53
0-24
004
004
0-63
0-40
0-45
0-36
ft
JJ
cell
8
0-34
0-25
0-24
014
ALA formed
(nM/ODU/h)
40
40
50
4-5
40
50
RNA
(ODU/108)
FL/1 R e / / /
RN
0-5
0-8
0-66
0-74
0-58
0-68
0-42
8
Table 1. 8-Aminolaevulinate synthetase activity in reticulocyte ly sates of prenatal FL\4 Re + j + and FL/1 Re f/f mice
m
m
n
ac
z
o
r
o
>
w
o
O
Os
Haem synthesis in f/f mice
311
S-Aminolaevulinate synthetase activity in pre-natal liver
Significant levels of ^-aminolaevulinate synthetase activity in lysates of FL/4
Re + / + foetal liver were found only during a restricted period of development
during days 13-14 of gestation, when the liver contains a relatively high proportion of early erythroblasts. Maximum values were obtained from foetal
livers of 9-10 mg wet weight which gave 50 pmol aminolaevulinate formed/
h/mg wet weight (i.e. 450-500 pmol aminolaevulinate formed/h/liver). Values
obtained from lysates of FL/1 Re///foetal livers from the 13—14th day of gestation were somewhat less (30 pmol aminolaevulinate formed/h/mg wet weight)
but detectable levels of activity continued into day 15 of development, although
at a lower level (14 pmol aminolaevulinate formed/h/mg wet weight). On the
13th, 14th and 15th day of gestation FL/1 Re///liver lysates formed aminolaevulinate at a rate equivalent to 300-350 pmol aminolaevulinate/h/liver.
The lower enzyme activity observed in///foetal liver is in proportion to the
reduced number of erythroblasts within///foetal livers during this period of
development (Bateman et al. 1972), and so does not necessarily indicate reduced
activity within individual///erythroblasts. The maintenance of enzyme activity
seen in / / / foetal livers is also correlated with the persistence of higher proportions of early erythroblasts in the liver which also characterizes this mutation.
('Normal' livers 35 % of early erythroblasts on day 13 falling to 20 % on day
16, fIf 50 % and 30 % respectively.)
^-Aminolaevulinate dehydratase activity in pre-natal reticulocytes
Levels of ^-aminolaevulinate dehydratase detected in circulating blood cells
of 17 to 18-day FL/1 R e / / / a n d FL/4 Re + / + foetuses are shown in Table 2.
Since there is little ^-aminolaevulinate dehydratase activity in mature erythrocytes (blood from young FL/4 Re mice with 1-2 % reticulocytes produced
300 nmol of porphobilinogen/1010 cells/h), comparisons of the two genotypes
may again best be made on the basis of number of reticulocytes to allow for
differences in the rates of maturation o f / / / a n d normal foetuses. When compared in this way, reticulocytes from///foetuses show approximately half the
^-aminolaevulinate dehydratase activity found in normal cells ( + / + 3476 +
506 nmol porphobilinogen produced/1010 cells/h, / / / 1788 +360 nmol/1010
cells/h).
^-Aminolaevulinate dehydratase activity in pre-natal liver
No differences in #-aminolaevulinate dehydratase levels were detected between///and wild-type foetal livers. On day 17, FL/1 Re///and FL/4 Re + / +
livers gave values of 1-7/tmol of porphobilinogen produced/g liver/h, similar
to that previously published (Coleman, Russell & Levin, 1969). On day 18
activity was reduced in livers of both genotypes to 1-0/tmol/g liver/h.
24-2
1
2
3
4
5
6
Expt.
1
2
3
4
5
6
Expt.
17
17
18
18
18
18
Means
(± standard
deviation of mean)
Foetal age
(days)
17
17
18
18
18
18
Means
(± standard
deviation of mean)
Foetal age
(days)
2213
1990
3185
1593
930
819
1788 ±360
PBG (nM/10
circulating
cells/h)
10
2132
3582
3950
2814
1896
1739
2686 ±347
PBG (nM/10
circulating
cells/h)
/o
100
100
100
100
100
100
retics
10
2345
3940
5530
3940
2655
2434
3474 ±506
PBG (nM/1010
retics/h)
4-2
3-9
5-6
5-6
4-6
4-6
ODU (RNA/
108 retics)
—
71
11-7
100
70
5-8
5-8
8-3
PBG (nM/
ODU RNA/h)
4-3
4-9
4-7
4-3
4-2
—
5-1
41
6-8
3-7
2-2
—
4-4
0-94
0-51
0-58
0-40
0-35
0-34
0-52
0-72
0-35
0-68
0-53
0-38
—
0-53
1-03
0-55
0-81
0-57
0-49
0-47
0-65
PBG/# retic. PBG/// RNA PBG/j/circulating cell
BPG/+ +
PBG/ + +
PBG/ + + circulating
ODU (RNA/
PBG (nin/
108 retics) ODU RNA/h)
retic.
RNA
cell
flf
90
90
70
70
70
70
retics
/o
+/+
Table 2. 8-Aminolaevulinate' dehydratase <activity in reticulocyte lysates of prenatal FLJ4 Re +/+ and FLjl Re f/f mice
tn
tn
X
o
M
>
o
r1
50
O
tn
n
o
oo
-«4
Foetal age
(days)
17
18
Fe incorporated
(pM/108/h)
15-5±0-8
4-6±10
Fe incorporated
(^M/ODU/h)
4-8
3-1
FL/4 Re + / +
3-2
1-5
19-8 ±5-0
9-5 ±1-6
17
J8
191 ±3-6
13-5 ±0-7
Fe incorporated
(pM/108/h)
/108
0-77
0
3-2
0
3-7
2-2
Fe incorporated
(pM/ODU/h)
FL/1 R e / / /
Fe incorporated
(PM/108/h)
12± 1-6
0
A
- Exogenous protoporphyrin
6-2
6-3
Fe incorporated
(pM/ODU/RNA/h)
RNA
(ODU/108)
RNA
(ODU/108)
Fe incorporated
(pM/108/h)
Foetal age
(days)
FL/1 R e / / /
FL/4 Re + / +
+ Exogenous protoporphyrin
/ODU
0-67
0
51
61
r
/108
0-97
1-4
/ODU
0-80
0-96
0-8
0-8
0-6
0
flf
protoporphyrin
+ + protoporphyrin
Fe incorporated
(pM/ODU/RNA/h)
Table 3. Haem synthetase activity in reticulocyte lysates of pre-natal FL/4 Re + / + and FLjl Re f/f mice
^-*5
S
380
R. J. COLE, J. GARLICK AND E. M. CHEEK
Table 4. Haem synthesis in actinomycin D-treated foetal liver cells in vitro
pM Fe incorporated into haem/h
FL/4Re+/+
Foetal
Labelling
age (days) period (hours) Control
12
13
13
3-5
3-5
4-6
0-6
0-6
2-9
FL/1 R e / / /
Actinomycin at
0 time
0-5
0-5
40
Control
0-76
0-59
0-8
Actinomycin at
0 time
10
0-43
0-9
Porphyrinogen synthetase in pre-natal blood
Circulating blood cells from 17 to 18-day FL/4 Re + / + and FL/1 R e / / /
foetuses show similar levels of porphyrinogen synthetase activity ( + / + 2597 ±
386 and///2905 ±472 nmol porphobilinogen consumed/h/1010 cells). However,
since no utilization of porphobilinogen was detectable in blood of young
FL 4/Re + / + mice with 1-2 % reticulocytosis, significant activity of this enzyme also appears to be restricted to reticulocytes. When expressed on the
basis of reticulocytes in the foetal circulation, 17 to 18-day///reticulocytes have
79% the activity of wild type ( + / + 3673 ± 585, fjf 2905 ± 472 nmol porphobilinogen consumed/h/1010 reticulocytes).
Haem synthetase activity in pre-natal reticulocytes
Levels of haem synthetase detected in circulating reticulocytes of 17 and
18-day FL/1 R e / / / a n d FL/4 Re + / + foetuses are shown in Table 3. When
excess protoporphyrin IX is present, 17-day cells o f / / / a n d + / + foetuses
show similar levels of incorporation of exogenous iron, compared on both a
'per cell' and 'per RNA unit' basis. Levels of activity in cells from 18-day
foetuses were considerably lower and more dependent on the availability of
exogenous protoporphyrin. With 18-day///reticulocytes, addition of protoporphyrin to the reaction mix appeared to be necessary for detection of any
incorporation of iron into haem.
Actinomycin D sensitivity of haem synthesis in pre-natal liver cells
Our results suggested that expression of the / / / genotype in later erythropoietic cells occurred only after the cessation of transcription, and might therefore be mediated by a differential effect on the stability of RNA species associated with haem synthesis. In order to test this possibility, freshly explanted foetal
liver cells were exposed to actinomycin D (4/^g/ml), and their subsequent
capacity to incorporate 59Fe into haem measured. Such cultures could not be
prolonged beyond 5-6 h without rapid loss of cells. Data presented in Table 4
Haem synthesis in f/f mice
381
indicate that blocking further transcription did not decrease haem synthesis in
///foetal liver cells within this period.
DISCUSSION
Previous studies on the enzymology of haem synthesis in flexed anaemic mice
have concentrated on the foetal liver at various stages of development (Coleman
et al. 1969). However, the cellular constitutions of livers of both///and +/ +
foetuses change rapidly during development so that activities expressed per
unit weight of liver give limited information on enzyme levels within the erythropoietic cells of the foetus. Data presented by Coleman et al. (1969) show that the
liver level of £-aminolaevulinate dehydratase rises by a factor of only 1-3 from
day 13 to day 16 in normal foetuses, although the proportion of erythroid cells
more than doubles during this period. These authors showed that #-aminolaevulinate dehydratase levels per unit weight of liver were comparable in + / +
and///foetuses throughout development.
In vitro studies on / / / and normal pre-natal erythroblasts (Bateman et al.
1972) indicated no significant difference in their ability to incorporate exogenous
59
Fe into haem. It is therefore unlikely that the hypochromia of pre-natal / / /
erythrocytes could arise from lesions in haem synthesis expressed during the
nucleated erythroblast stage of erythroid differentiation.
A number of studies have shown that a large proportion (•£-§) of the haemoglobin complement of erythrocytes derived from the foetal liver is synthesized
after extrusion of the nucleus at the orthochromatic stage (Fantoni, Chapelle,
Rifkind & Marks, 1968; Paul, Conkie & Freshney, 1969; Steiner & Vogel,
1973).
Microspectrophotometric determinations of haemoglobin content at various
stages of pre-natal erythroblast differentiation (Steiner, 1974) indicate that
1.6 pg haemoglobin, out of the total of 28 pg characteristic of each mature
liver-derived erythrocyte, are synthesized after the end of the orthochromatic
stage. This is equivalent to the accumulation after loss of the cell nucleus of
100 nmol of haem/108cells, dependent on formation of 400 nmol of porphobilinogen/108 cells and 800 nmol of #-aminolaevulinate/108 cells. Since the
maturation time of pre-natal liver-derived erythrocytes is approximately 24 h,
rates of porphobilinogen and haem formation achieved in cell-free systems
derived from normal pre-natal reticulocytes are comparable to those required
in vivo. However, aminolaevulinate formation is less than 10 % of that required in vivo, if it is assumed that the reticulocyte is 'self-supporting', i.e. that
there is no pre-formed pool of amino-laevulinate and no uptake from plasma.
Lesions expressed during the reticulocyte stage of erythroid maturation could
therefore have profound effects on the haemoglobin content of mature pre-natal
erythrocytes. Erythrocytes of neo-natal FL/1 R e / / / m i c e contain 1-7 mg
haemoglobin/108 cells, compared with 2-7 mg/108 FL/4 Re + / + cells, i.e. a
382
R. J. COLE, J. GARLICK AND E. M. CHEEK
reduction to two-thirds of the normal level. Therefore, if haem synthesis in
pre-natal nucleated///erythroblasts is normal, that in///reticulocytes must be
reduced to \-% normal to account for the observed hypochromia of mature
///erythrocytes derived from the foetal liver.
Previous studies (Cole et al. 1972) have shown that utilization of intracellular
iron for haem synthesis in / / / pre-natal reticulocytes is one-half that found in
normal cells and the present experiments show a reduction of similar magnitude
in #-aminolaevulinate synthetase and ^-aminolaevulinate dehydratase levels.
This observation is consistent with the recent finding (Kreimer-Birnbaum et al.
1972) that reticulocytes of 10-day FL/1 Re///"foetuses contain only 60 % of the
free protoporphyrin found in FL/4 Re + / + reticulocytes, per packed cell
volume. Since///erythrocytes are some 15 % smaller than + / + erythrocytes
the difference between///and + / + on a per cell basis will be nearly twofold.
Haem synthetase activity in homogenates of///prenatal reticulocytes is largely
dependent on added protoporphyrin while a more adequate preformed pool
appears to exist in normal pre-natal reticulocytes.
The reductions in aminolaevulinate synthetase and aminolaevulinate dehydratase activities observed in///reticulocyte homogenates is therefore quantitatively consistent with these lesions being a major contributory factor in
reduced pre-natal haemoglobin synthesis in this mutant.
It is generally accepted that #-aminolaevulinate synthetase is the most important rate-regulating step in haem synthesis in normal erythroid tissue,
although some regulation may also be exerted via ^-aminolaevulinate dehydratase (Calissano, Bonsignore & Cartasegna, 1966). Neither normal nor / / /
reticulocyte homogenates produced detectable levels of porphobilinogen unless
supplied with £-aminolaevulinic acid, so that it is unlikely that a significant
preformed pool of this precursor exists even in normal pre-natal reticulocytes.
Activities of ^-aminolaevulinate synthetase and ^-aminolaevulinate dehydratase
could therefore be rate-limiting steps restricting haem synthesis in reticulocytes
produced by / / / foetal livers. Both aminolaevulinate synthetase and aminolaevulinate dehydratase are subject to end-product inhibition by haem (Scholnick, Hommaker & Marver, 1972) and both are sulphydryl enzymes strongly
dependent on activation by cations. The synthesis of ^-aminolaevulinate
synthetase is repressed by haem, and also perhaps directly by aminolaevulinate
(Strand, Manning & Marver, 1972). Depressed activity of both enzymes in
pre-natal///reticulocytes could therefore be due to an underlying disturbance
of cytoplasmic homeostasis, e.g. ionic balance. However, ^-aminolaevulinate
synthetase usually occurs partitioned between the inner mitochondrial matrix
and its membrane (Patton & Beattie, 1973), while ^-aminolaevulinate dehydratase occurs in the 'cell sap'.
Alternatively, the primary lesion may be expressed as reduced activity of
(^-aminolaevulinate dehydratase, leading initially to a build-up of aminolaevulinic acid, and repression of formation of £-aminolaevulinicacid synthetase,
Haem synthesis in f/f mice
383
during the early erythroblast stage. Since the half-life of this enzyme is very
short, it is possible that subnormal levels could then be reached after the cessation of transcription as the erythroblasts mature to erythrocytes, so that
reticulocytes would show reduced activity of both enzymes, but for different
causes.
#-Aminolaevulinate dehydratase exists in at least two forms during development. The enzyme found in foetal liver is of higher activity, and different in
stability to heat and proteolytic inactivation than the enzyme found in adult
liver and spleen (Doyle & Schimke, 1969). Both forms of the enzyme are subject
to regulation of level by the Lv gene. No differences have been found in the
nature of the enzyme in livers or spleens between normal and / / / adult mice
(Coleman, 1966).
The flexed gene appears to have rather different effects in pre-natal and postnatal erythropoiesis. During pre-natal life the major effects, from which other
consequences flow, are a reduction in number and/or expression of haematopoietic colony-forming cells or their immediate descendants (Bateman & Cole,
1972) and reduced haem synthesis in reticulocytes. In adult///mice, enhanced
erythropoiesis in response to anaemia is delayed (Coleman et al. 1969), analogous
to the failure of pre-natal haemopoietic colony-forming cells in early foetal
liver, but the reticulocytes resulting from such enhanced erythropoiesis appear
to synthesize haem normally (Cole, Regan & Tarbutt, 1972). The phenotypic
lesions characteristic of the///gene complement therefore appear to be due to
disturbed synthesis of early precursors of haem which remain below threshold
levels necessary for normal development of early erythroid precursor cells in
both pre-natal and post-natal mice, and of reticulocytes in pre-natal mice.
Recognizable erythroblasts of both pre- and post-natal///haemopoietic tissues
and the reticulocytes of / / / post-natal mice appear to be able to function
adequately, suggesting either that these cell types can develop normally with
restricted rates of synthesis of haem precursors or alternatively that the molecular effects of the///lesion fail to be expressed in them.
The marked contrast between haem synthetic capacity of / / / pre-natal
erythroblasts, and that of enucleated reticulocytes derived from them, suggested
that a possible effect of the///gene complement could be to reduce the stability
of molecules (e.g. either RNA, or the enzymes themselves) associated with the
production of early haem precursors. Previous studies have shown that RNA
species within the liver erythroblasts of normal pre-natal mice, upon which
haemoglobin synthesis depends, do not become stabilized until the 14-15th
day of gestation (Fantoni et al. 1968), when characteristic changes in the cell
cycle parameters of erythroblasts also occur. However, haem synthesis in
explanted / / / pre-natal erythroblasts was no more sensitive to the effects of
actinomycin D than that in normal pre-natal erythroblasts (Table 4). These
different developmental stage- and cell-type-dependent patterns of gene expression of the/locus can be resolved by two assumptions: (1) that the effect of
384
R. J. COLE, J. GARLICK AND E. M. CHEEK
Decreas sd ALA ||
Repression of
etase Vr<- ALA synthetase
acti vity
||
production
Reduced utilization
of ALA
H
Decreased ALA
dehydratase
activity
Decreased protoporphyrin
synthesis
Retarded
foetal
growth
Decreased protein
synthesis
Failure of
coordination
of skeletal
growth
Decreased haem
synthesis
Hypochromia of
erythrocytes
Reduced proliferation
of haemopoietic
progenitor cells
Reduced production
of erythrocytes
Uncoordinated globin
synthesis and nonassimilation of iron
by reticulocytes
Reduced O2
tension in
foetal blood
Raised erythropoietin
levels in foetal
blood
i.
Shortened cell
cycle time
of liver
erythroblasts
Fig. 1. Suggested causal and circumstantial interrelations between phenotypic
expressions of mutation at the/locus in pre-natal mice. Data enclosed by double
lines are based on direct experimental observation.
/ l o c u s is restricted to forms of #-aminolaevulinate dehydratase and perhaps
#-aminolaevulinate synthetase characteristic of pre-natal erythropoietic tissues,
and (2) that this form of the enzyme(s) is retained by haemopoietic stem cells
(or early erythroid precursor cells) in post-natal spleen and bone marrow, but
replaced by the adult form of the enzyme as erythroid differentiation proceeds.
Haem is effective in promoting initiation of protein synthesis at the polysome
level for proteins other than haemoglobin and in non-erythroid cells (Matthews,
Hunt & Brayley, 1973; Beuzard, Rodvien & London, 1973; Raffel, Stein &
Kaempfer, 1974). Other characteristic phenotypic expressions of the flexed
locus, i.e. retarded pre-natal growth and reduced growth of particular skeletal
elements, could therefore also be due directly to restricted haem synthesis
caused by reduced activity of #-aminolaevulinate synthetase and £-aminolaevulinate dehydratase during pre-natal development in a variety of tissues. Since
congenitally anaemic SP/Sl1 foetuses grow at a normal rate (Cole, Tarbutt,
Cheek & White, 1974), although their pre-natal anaemia is more severe than
Haem synthesis in f/f mice
385
that of///animals, these phenotypic abnormalities of growth are unlikely to be
secondary to the anaemia. However, other anaemia mutants {SI and W series)
show abnormal pigment distribution similar to that in / / / and this expression
could be due to secondary effects of reduced tissue oxygenation.
#-Aminolaevulinate dehydratase is known to be particularly sensitive to lead
poisoning (Battistino et ah 1971). The possible dependence of spleen colonyforming cell multiplication and production of erythroid progenitor cells on
this enzyme, suggested here, indicates that lead poisoning may have critical
effects on primitive haematopoietic precursor cells, as well as on overtly
differentiating erythroid cells. In addition lead may affect the pre-natal growth
and differentiation of other tissues, e.g. skeletal elements, to cause permanent
structural defects via a primary effect on haem synthesis, so the teratogenic
effects of such interactions may be of considerable importance.
This work was supported by the Medical Research Council. We are grateful to Dr R. I.
Freshney (Cancer Research Department, Beatson Hospital, Glasgow) for advice on the assay
of haem synthetic enzymes.
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(Received 11 February 1975, revised 3 June, 1975)