Capitulo 13

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Developmental
Genetics
A. Ruvinsky
A. Ruvinsky
Developmental
13 andGenetics 1 and F. Stewart2
F. Stewart
1Animal Science, SRSNR, University of New England, Armidale,
NSW 2351, Australia; 2TBA Equine Fertility Unit, Woodditton Road,

Newmarket CB8 9BH and Developmental Genetics, The Babraham
Institute, Babraham, Cambridge CB1 2HN, UK
Introduction 344
Developmental Stages of the Horse Embryo 344
Genetic Control of Pre-implantation Development 348
Maternal regulation of early development 348
Genome activation 349
Embryonic gene expression 350
Trophoblast gene expression 353
Gametic imprinting 355
Maternal Recognition of Pregnancy and Placentation 358
Maternal recognition of pregnancy 358
Development of the placenta 358
Genes Involved in Control of Morphogenesis 360
Gastrulation 360
Notochord formation 361
Hox genes and development of axial identity 361
Organogenesis: T-box and Pax genes 362
Muscle development 364
Developmental effects of coat colour mutations 365
Sex Determination 366
The major steps in gonad differentiation 366
SRY gene and sexual differentiation 368
Cycle of the X chromosome 369
Sex reversals in the horse 370
Development of Interspecies Hybrids 371
Description of interspecies hybrids 371
Contribution of equine hybrids to developmental
genetics 371
References 373
©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky) 343
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344 A. Ruvinsky and F. Stewart
Introduction
Extensive investigation of mammalian development during recent years has
contributed significantly to a better understanding of developmental genetics
in general. However, the vast majority of information concerning mammalian
development has been generated through the use of mouse genetics. There-
fore, it may appear premature to write a chapter entirely devoted to develop-
mental genetics of the horse. On the other hand, there are a number of studies
on the embryology and genetics of development in the horse which, when
placed on a general background of mammalian development, could provide a
useful basis for future research in this area.
Despite the high level of similarity in mammalian development, there
are numerous contrasts between species resulting from their morphological
differences, placental structure, longevity and schedule of development. These
distinctive features of development based on genetic differences, which have
accumulated for tens of millions years of independent evolution, are still await-
ing clarification and understanding. Studying the differences often can be very
informative and we hope that gathering the available information on the horse
now will be a helpful reference and will provide a basis for future comparisons
and reviews on developmental genetics in this species.
Developmental Stages of the Horse Embryo

Gamete maturation and fertilization, which comprise the first crucial steps in
each new developmental cycle in mammals, have been considered in the
previous chapter. The embryological events and their genetic determination
which follow fertilization are discussed here. Table 13.1 summarizes the
essential events and timing of conceptus and fetal development in the horse.
Since fertilization occurs within several hours of ovulation (Thibault, 1967;
Morris et al., 2000) and ovulation can be timed accurately in the horse, days of
gestation are always counted from the day of ovulation. In this chapter, day 0
is taken as the day of ovulation and fertilization is assumed to occur within
24 h of ovulation. Three consecutive stages of development have been
defined: vesicular, embryonic and fetal (Table 13.1).
The vesicular (ovum) period covers the first 15–16 days after ovulation. It
is characterized by several crucial events including cleavage, morula formation
and compaction, blastocyst development, gastrulation and the start of extra-
embryonic mesoderm migration (Van Niekerk and Allen, 1975; Betteridge
et al., 1982; Bezard et al., 1989; Ginther, 1992). After compaction of the morula
at about day 4.5, the conceptus enters the uterus between days 6 and 7 after
ovulation and coincidentally begins to undergo blastocyst formation. Tight
intercellular junctions develop and this provides a condition for accumulation
of fluid within the central cavity (the blastocoele). The majority of external
cells of the blastocyst, called trophoblast or trophoectoderm, develop an
epithelial phenotype and are concerned with development of the amnion and
64
Developmental Genetics 345
Table 13.1. Essential events and timing of prenatal development in the horsea.
Stage of development Days after ovulation Cells/stage/crlb
Vesicular (ovum) period 0–15

Two-cell 1 2
Eight-cell 3 8
Genome activation 2–3 4–8
Morula compaction 4.5 ~16
Capsule formation 6 32
Entry into the uterus 5–6 16–32
Trophoblast differentiation 6–7 32–64
Blastocyst formation 6–7 64+
Hatching from zona pellucida 7–8 ~200
Migration of endoderm complete 8 200+
Embryonic disc 11–12 1000+
Loss of polar trophoblast 12 Thousands
Gastrulation (primitive streak) 13–14 Thousands
Mesoderm migration 13 Thousands
Notochord 14 Thousands
Maternal recognition of pregnancy 12–14 Thousands
First somite pair 15 Thousands
Embryonic period 16–40
Neural groove 16 14 somites
Head fold 18 16 somites
Closing of neural tube 18 16 somites
Vascularized yolk sac 19 18 somites
Beating heart and allantoic bud 21 24 somites
Visible limb buds 23 40+ somites
Capsule ruptures 22–23 40+ somites
Optic and otic vesicles visible 26–28 Vertebrae developing
Chorionic girdle formation 26–35 Vertebrae developing
Chorionic girdle invades 35–36 Vertebrae developing
Well-developed tail and elongating limbs 38 crl = 2 cm
Fetal period 40–term
Chorioallantoic placentation from 40 crl = 2.5 cm
Sex determinable 40 crl = 2.5 cm
Hooves 45–50 crl = 3–4 cm
Further development of limbs 60 crl = 6 cm
Eyelids close 60 crl = 6 cm
Head and neck in normal position 80 crl = 11 cm
First hair on lips 100 crl = 19 cm
Hair coat 270 Maturing fetus
Birth 320–340 (Considerable variation)
aCompiled from: Ewart (1897, 1915); Van Niekerk and Allen (1975); Betteridge et al. (1982);
Bezard et al. (1989); Cruz and Pedersen (1991); Ginther (1992); Bazer et al. (1993); Enders et al.
(1993); Guillemot et al. (1995); Hafez (1993); Jainudeen and Hafez (1993); Ménézo and Renard
(1993); Brinsko et al. (1995); Grondahl and Hytell (1996).
bcrl = crown–rump length.
65
placenta. The remaining cells, located at one pole beneath the polar tropho-
blast, form the embryoblast or inner cell mass (ICM). Later during gastrulation,
the ICM differentiates into the three primary germ layers of the embryo;
ectoderm, mesoderm and endoderm.
In addition to having a zona pellucida, the equine conceptus is enveloped
by an acellular capsule which starts to develop beneath the zona pellucida
at about day 6 after ovulation and disappears between days 22 and 23
(Betteridge et al., 1982). Therefore, when the blastocyst ‘hatches’ from its zona
pellucida between days 7 and 8, the capsule persists and expands along with
the conceptus so that, by about day 18, it has increased in mass approximately
20-fold, probably due to both fetal and maternal contributions (Oriol et al.,
1993). This embryonic capsule not only protects the conceptus physically but
is also thought to play a role in communicating with the mother and in
promoting the mobility of the conceptus which is important for transmitting
the maternal recognition of pregnancy signal (Bazer et al., 1986; Chu et al.,
1997; see below). The capsule also ensures that the conceptus remains spheri-
cal, and without it the equine conceptus would probably elongate as in the
ruminant species. By day 15, the conceptus is about 2 cm in diameter, the
embryonic disc and migrating extra-embryonic mesoderm are visible to the
naked eye and the first pair of somites have formed. Therefore, despite its
vesicular appearance and lack of placental attachment, embryogenesis is well
underway. We therefore define the embryonic period as beginning on day 16.
During the embryonic period (days 16–40), the yolk sac placenta forms
but, although the capsule disappears on about day 23, the conceptus remains
more or less spherical and it does not start to elongate significantly until after
day 40. Furthermore, although it lodges at the base of one or other of the uter-
ine horns at about day 18 and, when the capsule has disappeared, makes close
contact with the maternal endometrium, the true chorioallantoic placenta does
not start to form until about day 40. Nevertheless, embryogenesis proceeds at
a rate comparable with other large mammals, and essential morphogenetic
events, such as development of the head, vertebrae and appendages, occur, as
well as development of the nervous system, blood circulation and other major
internal organs. The most thorough description of early embryogenesis in the
horse can be found in Professor Cosar J. Ewart’s publications (Ewart, 1897,
1915). These papers, which contain detailed descriptions and drawings, have
been largely forgotten but they are well worth consulting. To give an example,
Fig. 13.1 shows a selection of drawings from his 1915 paper illustrating the
external features of a day 21 horse embryo. This paper concentrates on the
first 3 weeks and gives an accurate account of the development of the extra-
embryonic membranes, nervous system, sense organs, alimentary canal, heart
and blood vessels.
Sex determination also occurs during the late embryonic period, when the
crown–rump length of the embryo reaches about 2.5 cm. Furthermore, just
prior to transition into the fetal stage at day 40, a discrete portion of the
chorion known as the chorionic girdle (Ewart, 1897) invades the maternal
endometrium to form the so-called endometrial cups (Allen and Moor, 1972).
66
Fig. 13.1. Drawings of a 21-day horse conceptus and embryo (from Ewart, 1915). (A) Section
through the left uterine horn of the pregnant mare showing the intact, spherical conceptus in very
close contact with the endometrial lining of the uterus (approx. 75% of natural size). (B) Appear-
ance of the 11 mm embryo after removal of the chorion showing the vitelline veins (vv) and left
vitelline artey (va). (C) Side view of the embryo showing the right vitelline vein (vv), the amnion
(am), the branchial arches (ba), the fonto-nasal process (fp), the depression or stomodaeum (s)
which will form the mouth and the heart (ht). (D) Dorsal and ventral views of the embryo showing
20+ somites, the neural canal with its opening to the exterior at the caudal end (np), the amnion
(am) and the otic vesicles (ov).
67
These structures secrete large amounts of equine chorionic gonadotrophin

(eCG) into the maternal bloodstream (Cole and Goss, 1943) to ensure mainte-
nance of progesterone secretion from the maternal ovaries (Amoroso et al.,
1948). However, the endometrial cups are completely detached from the fetal
membranes and have generally died and been sloughed off the surface of the
endometrium by day 120 (Allen and Stewart, 1993).
The transition from embryo to fetus is generally considered to be when
organogenesis is complete and the taxonomic order of the embryo starts to
become identifiable through external features. In most species, this coincides
with establishment of the chorioallantoic placenta. Therefore, in agreement
with others (Allen et al., 1982; Ginther, 1992), we define the fetal stage as
beginning on day 40 and continuing to term (days 320–340). The first
significant event in the fetal period is that the expanding chorioallantois (or
allantochorion) starts to interdigitate with the endometrium to form the chorio-
allantoic placenta and thus takes over from the regressing yolk sac placenta.
The mature placenta of the mare is diffuse, epitheliochorial and micro-
cotyledonary in structure and it gradually elongates to completely fill the
body and both horns of the uterus (Samuel et al., 1974, 1976). Haemotrophic
nutrition takes place within the microcotyledons but histotrophic nutrition,
involving the absorption of endometrial gland secretions by columnar tropho-
blast cells within the inter-cotyledonary regions, also plays an important role
throughout gestation. Supplied by this placental nutrition, the fetus undergoes
numerous morphological changes and essential maturational events to prepare
it for postnatal life. While gestation length in mares normally ranges from 320
to 340 days, it varies considerably both within and between breeds, and viable
foals can be born as early as day 300 and as late as day 399 (Hintz et al., 1979;
Vanderplassche, 1980; Jainudeen and Hafez, 1993).
Comparing the developmental events and regulation of genes during
embryogenesis and fetal development in the horse with other farm mammals
can be useful (Cockett, 1997; Pomp and Geisert, 1998, Ruvinsky and
Spicer, 1999). In terms of placental development, the horse is most similar to
the pig and dromedary camel, both of which have an epitheliochorial
placenta.
Genetic Control of Pre-implantation Development

Maternal regulation of early development
There is considerable evidence in lower species that very early development is

controlled by maternal factors stored in or made by the oocyte (Gurdon, 1992;
St Johnston and Nüsslein-Volhard, 1992; Nüsslein-Volhard, 1996). This has not
been studied in the horse, but information from laboratory species has shown
that morphogen gradients also exist in the mammalian oocyte which are
believed to be important in specifying major polarities (Holliday, 1990; Fulka
et al., 1998).
68
Recent data indicate that the leptin and STAT3 proteins play critical roles
in early mammalian development, and may be involved in the determination
of the animal pole of the mammalian oocyte and in the differentiation of the
trophoblast and ICM (Antczak and Van Blekom, 1997). A potential role for
these proteins in early development is indicated at the morula stage where the
‘inner’ cells consist of blastomeres that contain little, if any, leptin/STAT3 while
‘outer’ cells contain both leptin/STAT3-rich and -poor cells (Antczak and Van
Blekom, 1997). It was also shown in the mouse that tropomyosin, an actin-
binding cytoskeletal protein, becomes associated both with the blastomere
cortex after fertilization and with the cleavage furrow during cytokinesis. The
interphase cortical association is uniform until the eight-cell stage, when
tropomyosin becomes associated with the developing apical pole and is
excluded from the basolateral cortex. Thus the early mouse conceptus con-
tains a unique and specific set of tropomyosins which respond to polarizing
signals (Clayton and Johnson, 1998).
Increasing cell polarity was described at the eight-cell stage in both the
mouse and rat (Gueth-Hallonet and Maro, 1992). Cell fate, controlled by
positional information, seems reversible and provides the developing embryo
with a certain degree of flexibility. In cattle, cell polarization has occurred in
some blastomeres at the eight- to 16-cell stage, but typical, distinct polarity is
not evident until after the 16-cell stage with approximately 40% polar cells per
embryo (Koyama et al., 1994)
Available data indicate a low level of embryonic gene expression in the
mouse during the first few cell divisions (Davis and Schultz, 1997). It is likely,
however, that replication of DNA and maintenance of the majority of cell
functions during the first two or three divisions is provided by RNAs and
proteins accumulated during oocyte maturation. oviducal proteins are also
believed to be involved. For example, oestrus-associated glycoprotein (EGP)
has been shown to influence early cleavage rate (Nancarrow and Hill, 1995).
The vast majority of transcripts studied from the one-cell stage onwards show
the same pattern of expression, with the number of copies per embryo declin-
ing from the one- to two-cell stage and then increasing dramatically (reviewed
by Kidder, 1993).
Genome activation
Electron microscopy studies suggest that activation of the horse embryonic

genome commences between the two- and four-cell stage (Grondahl et al.,
1993; Grondahl and Hyttel, 1996). In six- and eight-cell embryos, further
activation of the genome has been traced using [3H]uridine incorporation and
autoradiography (Brinsko et al. 1995; Grondahl and Hyttel, 1996). In 16-cell
embryos and beyond, transcriptionally active compact fibrillo-granular
nucleoli are evident (Grondahl and Hyttel, 1996), suggesting that nucleolar
activation, leading to the synthesis of rRNA and other nuclear RNAs, is initiated
during the fourth cell cycle. Figure 13.2 shows the poly(A) RNA profile during
69
Fig. 13.2. Dynamics of poly(A)+ RNA during the pre-implantation development of the mouse
embryo (from Ménézo and Renard, in Reproduction in Mammals and Man 1993, with permission
of Ellipses).
pre-implantation development in the mouse embryo. A similar picture is

expected for other mammalian embryos, including that of equids. The major
anticipated difference between species would be in time scale.
Little is known about development of the protein profile during early
equine embryogenesis, but comparative data obtained in other domestic and
laboratory mammals are probably applicable to the horse. Shehu et al. (1996)
showed that lamin B in bovine embryos appears as a constitutive component
of nuclei at all pre-implantation stages, whereas lamins A and C have a stage-
related distribution. The nuclei from the early cleavage stages contain lamins A
and C which generally disappear later, with a few possible exceptions in the
morula and blastocyst. Other proteins essential for morphogenetic events
appear in developing bovine embryos. These include several cytoskeletal and
cytoskeleton-related components such as F-actin, α-catenin and E-cadherin.
These proteins first appear on day 6 and their appearance and polarized
distribution are related to compaction of the morula (Shehu et al., 1996).
Data from the pig suggest that several more morphogenetically important
proteins appear during early cleavage and compaction, such as actin and the
actin-associated proteins α-fodrin, vinculin and E-cadherin (Reima et al., 1993).
These molecules are distributed evenly in blastomeres during early cleavage
but then accumulate gradually in regions of intercellular contact towards the
blastocyst stage (Reima et al., 1993).
Embryonic gene expression
Blastocyst formation creates two different cell lineages: non-polarized inner

mass cells and polarized trophectoderm or outer cells with prominent
70
microvilli. Louvet et al. (1996) showed in mice that this process is accompa-
nied by specific redistribution of the actin-associated protein ezrin, which is
proposed to play a role in the formation of microvillous structures that are
crucial for normal implantation. Before morula compaction, ezrin is located
around the cell cortex. However, after blastocyst formation, it segregates to
the outer trophectoderm cells which have microvilli. Two phosphorylated
forms of ezrin are present from the ovum period throughout pre-implantation
development, but they gradually decrease in amount. A third non-tyrosine-
phosphorylated isoform appears at the eight-cell stage and increases to the
blastocyst stage (Louvet et al., 1996). Several other actin-associated proteins
(α-fodrin, vinculin and E-cadherin), which are involved in cytokeratin bundles,
are not observed until the early blastocyst in both the mouse and the pig
(Reima et al., 1993). E-cadherin cell adhesion function is essential for the
establishment and maintenance of epithelial cell morphology during embryo-
genesis and adulthood. Mouse embryos homozygous for a targeted mutation
of the gene show severe abnormalities before implantation, because dissocia-
tion of adhesive cells of the morula has occurred shortly after compaction and
their morphological polarization is then destroyed. Maternal E-cadherin is able
to initiate compaction, but cannot maintain the process (Riethmacher et al.,
1995). Significant defects in the cell junctional and cytoskeletal organization
were found in E-cahedrin null mouse embryos, and the trophectoderm layer
failed to differentiate (Oshugi et al., 1997).
Although the rate of embryonic development is quite different in mice and
pigs, there is a close correlation between the developmental stage and
cytoskeletal organization in both species. Likewise, in the expanded bovine
blastocyst, the distribution of several cytoskeletal and cytoskeleton-related
proteins appeared similar (Shehu et al., 1996). Extracellular fibronectin was
first detected in the early blastocyst before differentiation of the primitive
endoderm, and at this stage was localized at the interface between the
trophectoderm and extra-embryonic endoderm (Shehu et al., 1996). Cingulin,
the tight junction peripheral membrane protein, also contributes to morpho-
logical differentiation in early mouse development and it is likely that other
mammals have the same gene. Its synthesis is tissue-specific in blastocysts, is
up-regulated in the trophectoderm and down-regulated in the ICM (Javed
et al., 1993).
It is commonly accepted that proto-oncogenes are involved in numerous
processes of embryonic development and that they encode a series of nuclear
transcription factors, intracellular signal transducers, growth factors and
growth factor receptors. For example, activation of the c-fos and c-jun proto-
oncogenes in sheep conceptuses occurs during the period of rapid growth and
elongation (Wu, 1996), and a similar pattern probably occurs in equine
embryos. These proto-oncogenes are involved in the regulation of gene
expression, cell proliferation and differentiation.
Information on the expression of housekeeping genes during equine
embryonic development is sparse but it is likely to be similar to that observed
in bovine embryos. The mRNA levels for various studied genes in bovine
71
embryos remain constant or decrease slightly from the mature oocyte to the
six- to eight-cell or morula stage and increase greatly in blastocysts. These
changes in gene expression were significant ranging from two- to sixfold to
110- to 118-fold (Bilodeau-Goeseels and Schultz, 1997). However, caution is
required when extrapolating from one species to another, particularly if the
species belong to different orders. For example, the equine embryonic exon 1
complementary DNA (cDNA) sequence from cytochrome P450 aromatase has
no significant homology with the corresponding region in porcine, human and
bovine sequences. This finding indicates divergence in the regulatory motifs of
this portion of the gene amongst mammals and suggests potential significance
with regard to activation of the ‘embryonic’ promoter and/or splicing of
novel aromatase 5 ′ exons in the transient production of oestrogens by
pre-implantaion blastocysts (Choi et al., 1996). A developmental switch in
expression from the blastocyst to endometrial/placental-type cytochrome P450
aromatase gene described in horses may function in embryo–maternal signal-
ling at the peri-implantation stage (Choi et al., 1997).
Two equine genes significantly activated between 12 and 15 days of
pregnancy were cloned recently. The first of them encodes calcyclin, which
belongs to the family of calcium-binding proteins. A similar protein was found
in mouse decidua and trophoblast. Expression of calcyclin increases approxi-
mately 30-fold from day 12 to 15. The other gene, that for phospholipase A2
(PLA2), is involved in release of arachidonic acid needed for prostaglandin,
thromoxane and leukotriene synthesis. Multiple transcripts of PLA2 were
detected and they appeared to be differentially regulated in day 12 and day 15
conceptuses (Simpson et al., 1999).
Many efforts have been made to try and ascertain the relative importance
of internal versus external factors in controlling the development of pre-
implantation embryos. Most experiments with rodents show that it is unlikely
that pre-implantation development is significantly dependent on external
factors. Furthermore, none of the known endogenously produced factors and
their receptors are essential until the blastocyst stage (Stewart and Cullinan,
1997). However, later during development, the importance of growth factors
increases dramatically.
It was found that some of the regulatory substances secreted by the
uterus can act as growth factors. Together with a number of growth factors
and their receptors produced by the embryo itself, they create the medium
essential for development. A detailed review of these regulators of mam-
malian embryonic development can be found elsewhere (Schultz and
Heyner, 1993). In equine embryos, transcripts for insulin-like growth factor
2 (IGF-2) were present at all examined stages (14–150 days). They were
found predominantly in tissues of mesodermal origin, but also in the
endoderm-derived liver and epithelia of the gut and lung bronchioles, and the
ectoderm-derived facial mesenchyme and choroid plexus (Lennard et al.,
1995a). Data suggest that the equine IGF2 gene is under developmental
control, with the possible existence of several promoters (Joujou-Sisic et al.,
1993).
72
As in all mammals, the equine ovum and early conceptus are protected by
a covering called the zona pellucida which is shed at blastocyst ‘hatching’. In
addition, as mentioned above, the equine zygote lays down another covering
beneath the zona pellucida, called the embryonic capsule, which is composed
of mucin-like glycoproteins. After hatching from the zona pellucida, the
capsule expands along with the equine conceptus but then appears to rupture
at about day 23; fragments of it have been seen as late as day 25 (Enders et al.,
1993). Therefore, in addition to surviving on the simple absorption of uterine
secretions during the first 3 weeks of pregnancy, these nutrients must
pass through the capsule. It is therefore not surprising that a number of
progesterone-dependent endometrial proteins have been observed in the
mare (Zavy et al., 1982; Beier-Hellwig et al., 1995; McDowell et al., 1995). One
of these, a novel 19 kDa protein has been characterized and shown to be a
member of the lipocalin family, most of which are transport proteins (Crossett
et al., 1996). This protein was discovered originally in the capsule of early
horse conceptuses (Stewart et al., 1995a) and later shown to be present in yolk
sac fluid up to day 20 of gestation (Crossett et al., 1998). The protein has not
yet been found in any other species, but homologues of it may exist and may
only be needed in small quantities for a short period of time. The reason that it
is so plentiful in the mare may be due to the presence of the capsule and the
need to transport a labile, maternal factor through it.
Trophoblast gene expression
Differentiation of trophoblast cells, the first differentiation event during

mammalian embryonic development, provides the key tissue for development
of the fetal–maternal interface during implantation and placentation. Reviews
of current knowledge about the genetic control of trophoblast development
and implantation (Cross et al., 1994; Rinkenberger et al., 1997; Schultz and
Edwards, 1997) are based largely on studies in mice. However, many features
of these processes are common for the majority of eutherian mammals and
to some degree are applicable to the horse. At present, 44 loci with a range
of functions have been implicated in pre- and peri-implantation events
(Rinkenberger et al., 1997).
A basic helix–loop–helix (bHLH) transcription factor gene, Hxt, later
named Hand1, is expressed in early trophoblast and in differentiated giant
cells of mouse embryos (Cross et al., 1995) and is essential for placentation
(Firulli et al., 1998; Riley et al., 1998). The negative HLH regulator, Id-1, inhib-
ited rat trophoblast (Rcho-1) stem cell differentiation and placental lactogen-I
transcription. These data indicate a role for HLH factors in regulating
trophoblast development and demonstrate a Hand1-positive function in pro-
moting formation of trophoblast giant cells. A preliminary study has demon-
strated very strong expression of the Hand1 gene in equine trophoblast cells,
particularly the chorionic girdle cells (F. Stewart and J.C. Cross, unpublished
data) and it is therefore almost certainly involved in the differentiation and/or
73
growth of equine trophoblast cells. A separate gene, Hed, encodes a related

protein that is expressed in maternal decidium surrounding the implantation
site (Cross et al., 1995). Another transcription factor gene, Mash-2, situated
in a locus homologous to the achaete/scute complex genes in Drosophila, is
also essential for successful placentation in mice. Its expression begins
during pre-implantation development but is restricted to the trophoblast
lineage after the blastocyst stage (Nakayama et al., 1997). This murine locus
belongs to the quite rare category of imprinted genes (Guillemot et al., 1995).
Mouse embryos which inherit a mutant Mash-2 allele from the mother and a
normal allele from the father die after implantation. The cause of death is a
lack of placental spongiotrophoblast (McLaughlin et al., 1996). The MMp9
gene, which is involved in development of giant trophoblast cells in mice
(Newman- Smith and Werb, 1997), is another candidate for an imprinted gene.
A specific form of imprinting manifests itself in the trophoblast of all species
studied whereby the paternally derived X chromosome is inactivated preferen-
tially in the trophoblast of female embryos (reviewed by Goto and Monk,
1998). Furthermore, a recent study involving targeted disruption of the
X-linked homeobox gene Esx 1, whose expression is restricted to extra-embry-
onic tissue, showed that expression of this gene from the maternal allele is
necessary for normal trophoblast morphogenesis in mice (Li and Behringer,
1998).
Genetic determination of integrin trafficking, which regulates adhesion to
fibronectin during differentiation of mouse peri-implantation blastocyst, has
been studied by Schultz et al. (1997). The regulation of several metalloprotein-
ase and corresponding genes may also shed additional light on the process of
implantation and further trophoblast development (Bass et al., 1997; Das et al.,
1997). However, since placental development in rodents is very different from
that in the horse, further research will be needed to determine if these murine
trophoblast genes are relevant to equine trophoblast development. A recent
study has identified an aspartic proteinase expressed by equine trophoblast
cells (Green et al., 1999). It is a member of the pregnancy-associated glyco-
protein (PAG) family and the horse is the first species outside the Artiodactyla
order (cattle, camels and pigs) in which a PAG has been identified.
Monoclonal antibodies raised against equine trophoblast cells have identi-
fied a number of potentially interesting equine trophoblast proteins (Antczak
et al., 1987). Two of the antibodies have identified proteins of 115 and 66 kDa,
respectively, in equine trophoblast tissue, and both antibodies stained human
trophoblast cells (Oriol et al., 1991). Significant efforts have also been made to
study developmental dynamics of class I and class II major histocompatibility
complex (MHC) antigens in trophoblast and endometrial cells in the horse.
The MHC class II antigens were not detected on any trophoblast cells, but
endometrial cells expressed them. On the other hand, it was found that MHC
class I antigens were expressed at high density on the surface of the tropho-
blast cells of the chorionic girdle at days 32–36, but these were down-regulated
as the cells invaded the maternal endometrium (Donaldson et al., 1990, 1992).
This phenomenon is considered to be involved in the mechanism which
74
prevents maternal immune rejection of the fetal–placental unit. The expression

of MHC class I genes may be controlled at the transcriptional level in horse
invasive and non-invasive trophoblast cells (Maher et al., 1996).
As expected, high concentrations of IGF-2 mRNA were detected not only
in the embryo itself but also in the extra-embryonic mesoderm, invasive
chorionic girdle and mature endometrial cup tissue (Lennard et al., 1995a).
Distribution of four cytokines in the endometrium and trophoblast of the horse
between days 30 and 55 of gestation showed that only tumour necrosis
factor-α (TNF-α) was present in the trophoblast cells. This cytokine might
have an important role in regulating trophoblast–endometrial interactions. The
other three cytokines studied, interleukin 2 (IL-2), interleukin 4 (IL-4), and
interferon-γ (IFN-γ) were not detected in trophoblast (Grunig and Antczak,
1995).
The terminally differentiated, trophoblast-derived, endometrial cup cells
secrete large quantities of the dimeric glycoprotein hormone, eCG (see
Chapter 12). There is now considerable evidence that the genes for the α- and
β-subunits of this hormone start to be expressed in the progenitors of the cup
cells, the chorionic girdle cells (McDowell et al., 1993; Wooding and Flint,
1994), and that their expression is linked to the cells becoming binucleate
and invasive. This is analogous to the situation in primates where the CG
genes are expressed in terminally differentiated syncytiotrophoblast. Equids
and primates are the only species which secrete a true chorionic gonado-
trophin during pregnancy, and both have a single glycoprotein hormone
α-subunit gene which is expressed in the pituitary gland and the tropho-
blast. However, unlike primates which have evolved a separate family of
trophoblast-specific β-subunit genes via duplication of an ancestral luteinizing
hormone (LH) β-subunit gene (Talmadge et al., 1984), equids express their LH
(pituitary) β-subunit gene in trophoblast cells (Sherman et al., 1992; Chopineau
et al., 1995). Thus, strictly speaking, equids secrete a placental (chorionic) LH.
Furthermore, unlike humans, and probably all primates, where CG is essential
for the recognition and maintenance of pregnancy, it does not appear to be
essential for pregnancy maintenance in equids (Stewart and Allen, 1995; see
Chapter 12).
Gametic imprinting
The fundamental assumption of Mendelian genetics is that the behaviour of

an allele is identical whether it arrives in a zygote through the paternal or
maternal germline pathway. Gametic imprinting phenomena discovered in
mammals show limitations of this classical view. Two sources of evidence
were essential to describe gametic imprinting. The first approach, based on
genetic evidence, demonstrated that some maternally and paternally derived
regions of certain chromosomes were not equivalent. Paternal or maternal
disomy of the regions containing particular genes caused significant effects on
viability and development of progeny (Lyon and Glenister, 1977; Cattanach,
75
1986). The second set of data was obtained by nuclear transplantations and
parthenogenetic activation of mammalian oocytes. These data showed that the
contribution of parental genomes was not equivalent, and differential imprint-
ing of nuclear genes during gametogenesis was very likely (McGrath and
Solter, 1983; Surani et al., 1984). Until now, the main bulk of information
regarding imprinting comes from the mouse and to a lesser extent from human
studies (Barlow, 1995). The number of loci found in mice showing gametic
imprinting currently is over 35 (Beechey and Cattanach, 1999).
Gametic imprinting is generally viewed as a mammalian phenomenon and
there are differences in imprinting patterns between species. The developmen-
tal function of gametic imprinting is still under intensive investigation, but an
explanation proposed by Moore and Haig (1991) is widely spread. It is based
on the idea of involvement of imprinted genes in the control of fetal growth
and fetal–maternal interactions, thus keeping a balance between contradictory
fetal and maternal requirements. It is therefore possible that gametic imprint-
ing evolved in mammals to regulate intrauterine growth to ensure a safe
outcome of pregnancy.
Details of the molecular mechanisms responsible for gametic imprinting
are not entirely understood, but in several instances it has been shown that
imprinting ‘marks’ are imposed on the control regions of imprinted genes
during gametogenesis by a parent-specific DNA methylation process (Shemer
et al., 1996; Bartolomei and Tilghman, 1997). These marks are resistant to
global demethylation during cleavage and de novo methylation after
implantation and also maintain different methylation patterns in the paternal
and maternal alleles of imprinted genes (Solter, 1998).
Acquisition of imprints is believed to occur before fertilization and imprint
propagation takes place until the morula–blastocyst stage. It seems likely,
however, that primary gametic signals are not simply copied from the gametes,
but rather methylation patterns typical for imprinted genes are established
gradually during early development (Shemer et al., 1996). Gametic imprinting
is likely to have evolved in mammals by adopting already existing epigenetic
mechanisms. The latest data indicate that imprinting in mammals and gene
silencing in Drosophila may have some similarities (Surani, 1998). Imprinting is
a reversible phenomenon and can be achieved only if erasure of imprinting
signals occurs in each consecutive developmental cycle. The erasure occurs in
primordial germ cells and, soon after that, new epigenetic modifications occur
at specific CpG nucleotides in imprinted genes (Goto and Monk, 1998; Surani,
1998; Ruvinsky, 1999).
Data on farm animals are still limited and include some indirect evidence
of gametic imprinting (reviewed by Ruvinsky, 1999). A recent comparative
study of normal and parthenogenetic embryos in sheep is the first direct
evidence that in sheep, as in mice and humans, the growth-related PEG1/Mest
and IGF2 genes are expressed from the paternal alleles (Feil et al., 1998).
Thus, there are convincing indications that gametic imprinting is a common
phenomenon in mammalian species, including equids.
76
A study of eCG concentration in the serum of pregnant mares and

jenny donkeys carrying intraspecies and interspecies embryos indicated that
production of the hormone is influenced by gametic imprinting (Allen et al.,
1993). It was suggested that differential expression of maternal and paternal
alleles may control the size and secretory activity of the fetal endometrial
cups, the structures that secrete eCG. However, embryo bisection to create
identical twin embryos and their subsequent transfer to different species
showed that any such effect was overridden by the maternal uterine environ-
ment (Allen et al., 1993). Nevertheless, imprinting of, for example, the IGF2
gene, may contribute to placental and/or fetal growth effects in mules and
hinnies. Similar phenomena were observed in mice (reviewed by Ruvinsky,
1999).
The equine IGF2 gene recently has been cloned and characterized
(Otte et al., 1998). It spans a 9 kb region, which is substantially less than the
corresponding human gene. Three coding exons and three untranslated leader
exons, all highly homologous to those in other species, were identified. Down-
stream of the polyadenylation site in exon 6, a dinucleotide repeat sequence
was identified. Three putative promoters (P1–P3) were localized in the 5 ′
region of the gene. RNase protection analysis revealed two active promoters in
fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult
tissues. This represents a transcriptional pattern different from that in humans
and rodents. A novel structural element, an inverted repeat, is predicted in the
3 ′ region of the IGF2 gene. This repeat is conserved between species and
located in a region which is differentially methylated in the human and mouse
genes and might therefore be involved in the imprinting mechanism. The
inverted repeat acquires a stem–loop structure in vitro with a hybrid A/B-DNA
conformation in the stem area.
In both horse and mouse, a methylation-sensitive protein binds this
structure with a strong requirement for the loop area. Furthermore, the protein
might be developmentally regulated (Otte et al., 1998). The IGF2 gene was
mapped to ECA12q13, which appears to be homologous to human chromo-
some 11 (Raudsepp et al., 1997) and murine chromosome 7. Interestingly this
gene tends to maintain a terminal location on the chromosome arm in a
number of mammalian species.
Lack of maternally or paternally derived alleles in a zygote causes, in
several instances, embryonic mortality and should therefore impose strict
requirements on the stability of imprinting signals. Successful cloning of
mammals using somatic cells of adult individuals (Wilmut et al., 1997) is the
first evidence of stability of differential imprinting signals maintained in
somatic cells long after intrauterine development.
It is possible that knowledge about the influence of the pathway (paternal
or maternal) used by an allele to enter the next generation will be adopted
sooner or later by selection programmes. Selection of modifier genes may
significantly change the effect of gametic imprinting, and this information
should also be taken into consideration.
77
Maternal Recognition of Pregnancy and Placentation

Maternal recognition of pregnancy
The mare must recognize the presence of the embryo in her uterus before day
12 after ovulation in order to prevent luteolysis of the primary corpus luteum
(CL). If she fails to do so, the CL will have regressed completely by day 16,
thereby withdrawing the progesterone support needed for pregnancy. As in
most species, the mechanism by which luteolysis is prevented in equine
pregnancy involves suppression of the release of prostaglandin F2α (PGF2α)
from the uterus. The embryonic signal(s) which prevent this release have not
been characterized fully in the mare, but several candidate factors, based on
studies in other species, have been investigated (Bazer et al., 1994).
In the pig, which has a similar type of placentation to the horse, the anti-
luteolytic signals include embryonic oestrogens and the equine conceptus has
been shown to release significant amounts of oestrogens between days 10 and
30 (Heap et al., 1982; Zavy et al., 1984). However, attempts to prolong the life
span of the CL in the mare by injecting oestrogens have provided inconclusive
results (Goff et al., 1993).
It has now been established that conceptus-derived interferon-tau (IFN-τ)
constitutes the major recognition signal in most ruminants (Bazer et al., 1994).
However, repeated attempts to find an equivalent gene or protein in the horse
have failed (Roberts et al., 1992; see Chapter 12) and embryonic oestrogens
remain the front running, although as yet unproven, luteostatic mechanism in
the pregnant mare.
One reason why it has proved difficult to identify a specific embryonic
factor in the mare may be the presence of the embryonic capsule. Most studies
to date have excluded the capsule, yet it is this structure that is in closest
contact with the maternal endometrium. The embryonic signals must pass
through the capsule and this may involve interactions and/or modifications
which are essential for correct presentation and/or interaction with the
endometrium. Mobility of the equine conceptus within the uterine lumen is
certainly important in the signalling process as restricting this movement
results in pregnancy failure (McDowell et al., 1988). In addition to the primary
CL, the pregnant mare develops several accessory CLs in her ovaries in
response to the eCG produced by endometrial cups. However, although these
help to maintain elevated maternal blood levels of progesterone until the
placenta takes over the role between days 80 and 100 (Holtan et al., 1975),
secondary luteal development is not absolutely necessary (Allen and Stewart,
1993; see Chapter 12).
Development of the placenta
Placentation in the mare is a prolonged process, involving a transient yolk sac

placenta, which becomes fully functional from about day 23 when the capsule
78
has disintegrated, and the gradual development of the chorioallantoic placenta

from around day 40. The time of implantation is therefore very difficult to
define in the mare and is considered by some to begin at about day 25, and by
others on day 40. Furthermore, since the equine conceptus does not ‘implant’
in the true sense, the term implantation is probably best avoided in this
species. Equine placentation is also characterized by the development of the
unique chorionic girdle and its invasion of the maternal endometrium to form
the gonadotrophin-secreting endometrial cups (see Chapter 12).
There have been few studies on genes that control development of the
yolk sac, but gene knock-out experiments in mice have demonstrated
profound effects of removing fibronectin (George et al., 1993), vascular endo-
thelial growth factor (VEGF; Carmeliet et al., 1996; Ferrara et al., 1996), VEGF
receptors (Fong et al., 1995; Shalaby et al., 1995), transforming growth
factor-β1 (TGF-β1) (Dickson et al., 1995) and the GATA transcription factors
(Koutsourakis et al., 1999). Although these factors have not been studied in
the horse, they are very likely to be involved in establishing a functional,
vascularized yolk sac placenta in this species.
In eutherian mammals, the allantois fuses with the chorion to form the
allantochorion. The allantois first apppears in the equine conceptus at
around day 22. Unlike humans and rodents, it is fluid filled and lined
with endoderm so that the equine allantochorion is composed of three
layers; trophoblast, vascularized allantoic mesenchyme and endoderm. Fusion
of the chorion with the allantoic mesenchyme is a crucial step in all
species and, in mice, it has been shown to depend on the expression of
vascular cell adhesion molecule-1 (VCAM-1) (Gurtner et al., 1995; Kwee et al.,
1995) and α4 integrin (Yang et al., 1995). By day 25, the allantoic sac of the
still-spherical equine conceptus is about one-third the size of the yolk sac
and, by day 30, the two sacs are more or less equal in size. It is during this
period that the chorionic girdle develops on the surface of the conceptus,
precisely where the underlying yolk and allantoic sacs abut one another.
The trophoblast cells in the girdle region multiply rapidly and pile up on
one another to form a 3–4 mm white band of tissue which encircles the
entire conceptus. The mechanisms that control the development of this
equine-specific structure are not entirely clear, but they must depend largely
on local, fetally derived mitogenic signals. The allantoic mesenchyme is the
most likely source of these signals (Stewart, 1996) and expression of two such
signals, IGF-2 and hepatocyte growth factor–scatter factor (HGF-SF), has
indeed been identified in horse allantoic mesenchyme (Lennard et al., 1995a;
Stewart et al., 1995b). HGF-SF is probably the more important of the two,
since both HGF-SF and HGF-SF receptor (c-met) knock-out mice fail at
around mid-gestation due to a major placental defect involving the lack of
labyrinthine trophoblast (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al.,
1995). Maternal growth factors, such as TGF-β1 (Lennard et al., 1995b) and
epidermal growth factor (EGF) (Lennard et al., 1998), may also be involved in
chorionic girdle formation but are likely to play only a permissive role in the
process.
79
After the chorionic girdle has detached from the fetal membranes to form
the endometrial cups, the allantochorion at last begins to interdigitate with the
maternal endometrium to form, eventually, the very complex microvillous
structure that is needed to ensure fetomaternal exchange until term. Both fetal
and maternal mitogenic and morphogenic factors obviously continue to be
important during this process.
Genes Involved in Control of Morphogenesis

Gastrulation
Gastrulation in the horse starts on days 13–14 of development (Ginther, 1992).

Cell proliferation and rearrangement in the germinal disc are the main events
during gastrulation in eutherian embryos. The visible indication of com-
menced gastrulation in the horse conceptus is formation of the primitive
streak. In mammals, ‘this process begins with the production and proliferation
of mesodermal progenitor cells at the proximal (allantoic) end of the primitive
streak; this position marks the future caudal end of the fetus. As ectodermal
cells migrate through the primitive streak, they move both laterally and distally
towards the future cranial end of the embryo, extending the primitive streak
towards the distal lip’ (Wilkins, 1993). A white peripheral zone, which can be
seen encircling the germinal disc at day 14 in the horse, is mesoderm growing
beneath the trophoblast layer (Van Niekerk and Allen, 1975; Ginther, 1992).
While genetic mechanisms that are responsible for gastrulation in mammals
are still mainly unknown, new data are starting to arrive (Viebahn, 1999). The
next step, establishing anterior–posterior orientation, recently became the
subject of intensive investigations (Beddington and Robertson, 1998). Two
genes, the homeobox gene goosecoid (gsc) and the winged-helix gene hepatic
nuclear factor-3β (HNF-3beta) are co-expressed in all three germ layers in
the anterior primitive streak and at the rostral end of mouse embryos during
gastrulation (Filosa et al., 1997). Fgf-4, a member of the fibroblast growth
factor (FGF) gene family which shows expression in the primitive streak and
a sequential expression in developmental pathways such as mesoderm
formation and myogenesis, is believed to play a role in specific epithelial–
mesenchymal interactions (Niswander and Martin, 1992).
The recently discovered murine Axin gene seems to be a crucial regulator
in embryonic axis formation in vertebrates. This gene inhibits the Wnt/
Wingless signalling pathway, involving several polypeptides and enzymes
(Zeng et al., 1997). This pathway plays an important role not only in embry-
onic development but also in tumorigenesis. Interaction of axin protein with
glycogen synthase kinase-3β is required for β-catenin down-regulation.
β-Catenin and axin are positive and negative effectors of the Wnt signalling
pathway, respectively (Nakamura et al., 1998).
The T gene, which is required for extension of the posterior axis
(Clements et al., 1996) and for several other essential steps in mammalian
80
development, including notochord formation, is discussed below. The next

step in development is the so-called ‘head process’, which gives rise to the
notochord and contributes to part of the endodermal lining of the gut.
Notochord formation
The notochord is a rod-shaped structure which extends along the embryo and
represents the initial axial skeleton, playing an important role in induction of
the neural plate, chondrogenesis and somite formation (Gomercic et al., 1991).
Early development of the notochord in the equine embryo has not been stud-
ied, but is estimated to begin at about day 14 after ovulation. Its histological
appearance in the embryo at day 21 is described in detail by Ewart (1915) and
also in a three-dimensional reconstruction of Ewart’s embryo (Robinson and
Gibson, 1915).
Clearly, activation of nuclear genes responsible for basic morphogenetic
rearrangements is requisite for notochord formation and development. The T
gene, which was first described as the Brachyury mutation in mice 70 years
ago, is an important participant in events required for differentiation of the
notochord and formation of mesoderm during posterior development. The T
protein is located in the cell nuclei and acts as a tissue-specific transcription
factor (Kispert et al., 1995). Cloning and sequencing of the T gene led to the
discovery of the T-box gene family, which is characterized by a conserved
sequence, called the T-box (Bollag et al., 1994). This ancient family of tran-
scription factors which underwent duplication around 400 million years ago is
common to all vertebrates (Ruvinsky and Silver, 1997). There are indications
that several murine T-box genes are essential in different mesodermal
subpopulations and one is essential in early endoderm during gastrulation
(Papaioannou, 1997). Involvement of the T-box genes Tbx2-–bx5 in vertebrate
limb specification and development was shown recently (Gibson-Brown et al.,
1998). Formation of the notochord leads to several key ontogenetic events
including induction of the neural tube and development of the gut, heart and
brain. A putative morphogen secreted by the floor plate and notochord, Sonic
hedgehog (Shh), specifies the fate of multiple cell types in the ventral aspect of
the vertebrate nervous system. Shh, in turn, induces expression of the onco-
gene Gli-1, which affects later development of dorsal midbrain and hindbrain
(Hynes et al., 1997).
Hox genes and development of axial identity
The homeotic genes, which encode transcription factors, were first described
in Drosophila as the primary determinants of segment identity. They all contain
a similar 180 bp DNA sequence motif named the homeobox. Comparative
analysis of the Drosophila homeotic gene complex called HOM-C and the
mammalian (murine) homeobox genes called the Hox complex demonstrates
81
a striking case of evolutionary conservation. The Hox gene family determines a

set of transcription factors crucial for development of axial identity in a wide
range of animal species (Maconochie et al., 1996). Figure 13.3 shows the
striking similarity and colinearity found in the molecular anatomy of the insect
and mammalian (vertebrate) Hox complexes. The main difference is the num-
ber of complexes per genome. In insects, there is only one, while mammals
and other vertebrates have four paralogous sets of genes.
Although only two homeobox genes belonging to the H6 homeobox gene
family, HMX1 and HMX2, have been studied in the horse (Stadler et al., 1995),
they both show a high degree of homology to the equivalent genes in other
species. This suggests that the main features of the murine Hox complexes
would be typical for the homologous equine gene complexes.
The Hox genes are expressed in a segmental fashion in the developing
somites and central nervous system, and each Hox gene acts from a particular
anterior limit in a posterior direction. The anterior and posterior limits are
different for different Hox genes (Fig. 13.3). The genes located at 3 ′ end have
the most anterior limit of activity. Transcription of the genes, however, moves
in the usual 5 ′–3 ′ direction. The genes located at the 3 ′ end are expressed
earlier and genes located at the 5 ′ end are expressed later. The process of
segmentation moves along the anterior–posterior axis, but there are differ-
ences in development of segmentation between the hindbrain and the trunk
(Maconochie et al., 1996). Thus, the vertebrate body is, at least partially, a
result of interactions of Hox genes that provide cells with the essential posi-
tional and functional information. Signals from the Hox genes force embryonic
cells to migrate to the appropriate destination and generate certain structures.
Retinoids can affect the expression of Hox genes, and there is a 5 ′–3 ′ gradient
in responsiveness of Hox genes to retinoids (Marshall et al., 1996).
A key role for the neural crest as the source for numerous cell lineages,
including sensory neurons, glial cells, melanocytes, some bone and cartilage
cells, thyroid cells and smooth muscle, is well known (Le Douarin, 1982).
There has been considerable progress during the past few years in identifying
genes controlling development of the neural crest and associated cell migra-
tion (Anderson, 1997). Several growth factors affect the developmental fate of
neural crest cells: glial growth factor (GGF), TGF-β which promotes smooth
muscle differentiation, and a bone morphogenic protein (BMP2/4) involved in
bone morphogenesis. Transcription factors are also important in neural crest
lineage determination, including the bHLH transcription factors Mash1 and
Mash2 (Anderson, 1997).
Organogenesis: T-box and Pax genes
Some of the T-box genes are involved in limb morphogenesis and specifica-
tion of forelimb/hindlimb identity. It was shown that Tbx5 and Tbx4 expres-
sion is restricted primarily to the developing fore- and hindlimb buds, respec-
tively. These two genes appear to have been selected divergently in vertebrate
82
Fig. 13.3. (A) Alignment of the four mouse Hox complexes with that of HOM-C from Drosophila.
The vertical shaded boxes indicate related genes. The 13 paralogous groups are noted at the
bottom of the alignment. The colinear properties of the Hox complexes with respect to timing of
expression, anteroposterior (A-P) level, and retinoic acid (RA) response are also noted at the
bottom (from Maconochie et al., 1996, with the author’s permission). (B) Summary of HOM-C
and Hox-2 expression patterns. The upper part of the figure is a diagram of a 10 h Drosophila
embryo with projections of expression patterns of different genes from the HOM-C complex to
particular body segments. The lower part of the figure is a diagram of a 12 day mouse embryo
with projections of expression patterns of different genes from the Hox-2 complex to particular
body segments (from McGinnis and Krumlauf, 1992, with the author’s permission).
83
evolution to play a role in the differential specification of fore- (pectoral)

versus hind- (pelvic) limb identity (Gibson-Brown et al., 1998). Mutations in
the human TBX3 gene cause the ulnar–mammary syndrome characterized by
posterior limb deficiencies or duplications, mammary gland dysfunction and
genital abnormalities. It was suggested that TBX3 and TBX5 evolved from a
common ancestral gene and each has acquired specific yet complementary
roles in patterning the mammalian upper limb (Bamshad et al., 1997).
Pax genes are another family of developmental genes encoding nuclear
transcription factors. They contain the paired domain, a conserved amino acid
motif with DNA-binding activity. Pax genes are key regulators of development
in organs and structures such as the kidney, eye, ear, nose, limb muscles,
vertebral column and brain. Vertebrate Pax genes are involved in pattern
formation possibly by determining the time and place of organ initiation or
morphogenesis (Dahl et al., 1997). Pax-1, for instance, is a mediator of noto-
chord signals during the dorsoventral specification of vertebrae (Koseki et al.,
1993). The Pax-3 gene may mediate activation of MyoD and Myf-5, the
myogenic regulatory factors, in response to muscle-inducing signals from
either axial tissues or overlying ectoderm and may act as a regulator of somatic
myogenesis (Maroto et al., 1997). Mutations in the Pax-2 gene prove
involvement of this gene in eye formation, as mutations in Pax-6 result in eye
malformation, known as aniridia in humans and small eye syndrome in mice,
(Dahl et al., 1997). Aniridia in horses, which has been described many times in
several breeds, may also be caused by changes in the homologous gene (Joyce
et al., 1990; see Chapter 4). The eyes absent gene (Eya2), which is involved in
eye development in several metazoan phyla, may also be relevant to horse
development. Like the Pax-6 gene family, Eya2 was probably recruited for
visual system formation some considerable time after its original function was
established (Duncan et al., 1997). Several other genes, such as Bmp-4, Msx-1
and Msx-2, which encode bone morphogenetic proteins and are expressed
before and after neural tube closure, interact with Pax-2 and Pax-3 (Monsoro-
Burq et al., 1996).
Muscle development
Information on development of muscle tissue in the horse is also important

from a practical point of view. Firulli and Olson (1997) have reviewed recent
progress relating to genetic mechanisms of muscle development in mammals.
Skeletal, cardiac and smooth muscle cells express overlapping sets of muscle-
specific genes, although some genes are only expressed in one particular
muscle type. So-called genetic modules or independent cis-regulatory regions
are required to direct the complete developmental pattern of expression of
individual muscle-specific genes within each muscle type, and the temporo-
spatial specificity of these regulatory modules is established by unique combi-
nations of transcription factors (Firulli and Olson, 1997). A gene encoding an
actin-modulating protein, gelsolin, from equine smooth muscle recently was
84
cloned and studied (Koepf et al., 1998). Comparison with human gelsolin has
shown a high degree of identity (94–95%). The same observation is true for
several other compared mammalian species.
It is well established that mitogens inhibit differentiation of skeletal muscle
cells, but the IGFs, acting through a single receptor, stimulate both prolifera-
tion and differentiation of myoblasts. For example, an inhibitor of mitogen-
activated protein (MAP) kinase inhibits IGF-stimulated proliferation of L6A1
myoblasts and associated events, such as phosphorylation of the MAP kinases
and elevation of c-fos mRNA and cyclin D protein. This inhibitor caused a
dramatic enhancement of differentiation, evident at both a morphological and
a biochemical level. In sharp contrast, an inhibitor of phosphatidylinositol
3-kinase and p70 S6 kinase completely abolished IGF stimulation of L6A1
differentiation. These data demonstrate that the MAP kinase pathway plays a
primary role in the mitogenic response and is inhibitory to the myogenic
response in L6A1 myoblasts, while activation of the phosphatidylinositol
3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation.
Thus, it appears that signalling from the IGF-1 receptor utilizes two distinct
pathways leading to either proliferation or differentiation of muscle cells
(Coolican et al., 1997).
Selection for greater muscle mass in horses may use some mutations
affecting muscle development. Hyperkalaemic periodic paralysis is the result
of one such mutation in the sodium channel gene, which is expressed in skele-
tal muscle. It is inherited as an autosomal dominant trait. The classical signs of
this syndrome are muscle fasciculation, spasm, and weakness associated with
hyperkalaemia (Naylor, 1994, see Chapters 4 and 8).
Developmental effects of coat colour mutations
Classical coat colour genetics in mammals has acquired developmental and

molecular orientation (Jackson, 1994). Most data were obtained in mice, but
the high homology of mammalian genomes provides a sufficient foundation
for extension to other species including the horse (see Chapter 3). Colour
mutations give excellent examples of numerous pleiotropic effects. One of the
reasons for this phenomenon is that mutations of several coat colour loci affect
normal development of the neural crest region, which plays a key role in
migration of melanoblasts, neuroblasts and other cell types (Anderson, 1997).
Details about migration of melanoblasts and neuroblasts in the horse are
unknown but it probably occurs within a week or so after day 18.
A recent molecular genetic analysis of Overo lethal white syndrome
(OLWS) in horses connected this dominant autosomal disorder with a muta-
tion in the endothelin receptor B (EDNRB) gene and neural crest migration
(Santschi et al., 1998). It was found that a substitution in codon 118 of
the equine EDNRB gene changes the characteristics of the transmembrane
domain 1 of a seven transmembrane domain G-protein-coupled receptor. This
mutation causes not only white coat colour in homozygous foals, but also
85
intestinal aganglionosis (Hultgren, 1982) which results in a poorly developed

enteric nervous system and rapid death of the foal soon after birth. It is most
likely that disturbances in neural crest cells caused by this mutation explain
this phenomenon in horses. Similar syndromes in humans and mice have been
described and induced in mice by mutation in the same gene (McCabe et al.,
1990).
Mutations which affect melanocyte morphology and create dilute colours
are common in mammals. For example, it was shown that mutations in a
myosin protein (Jackson, 1994), which may be caused by a proviral insertion
(Jenkins et al., 1981), led to lack of dendrites in melanocytes and diluted coat
colour. Interestingly, neuronal dendrites are not affected by these mutations.
Several other mutations affect melanogenic enzymes and related proteins.
Mutations in the tyrosinase gene lead to albino phenotype, while brown
colours, at least in mice, are the product of mutations in a locus encoding a
tyrosinase-related protein (Jackson, 1994). Different pleiotropic effects of these
mutations, including decreased viability, have also been observed.
Mutations of two other coat colour loci, agouti and extension, affect regu-
lation of melanogenesis. It was shown that the ratio between black eumelanin
and yellow phaeomelanin is regulated by α-melanocyte-stimulating hormone
(αMSH). The product of the agouti gene is an antagonist of αMSH and the
extension gene encodes the αMSH receptor. Again, there are several develop-
mental effects of mutations in these loci including obesity and tumour forma-
tion. The chestnut coat colour in different horse breeds which is controlled by
a recessive allele in the extension locus (e/e) is the result of a mutation in the
MSH receptor gene (MC1R). A missense mutation at codon 83 (TCC→TTC)
which causes a non-conservative substitution (Ser→Phe) in the MCIR protein
is associated with the e allele (Marklund et al. 1996). This mutation probably
alters the α-helix structure of the second transmembrane domain of the pro-
tein, leading to a defect in the receptor. A similar mutation has been described
in humans (Valverde et al., 1995). Obviously molecular analysis of coat colour
mutations provides good opportunities for a greater understanding of basic
developmental processes.
Sex Determination
The major steps in gonad differentiation
The earliest stages of gonadal development in mammals occur at a similar

stage in XX and XY embryos. Primordial germ cells, which differentiate
relatively late in mammals, migrate into the gonads of either presumptive sex
indiscriminately and may function even across a species barrier (McLaren,
1998, 1999). To be functional, a gonad needs both germ cells and somatic
cells. Assuming that gonadal development in the horse does not deviate
strongly from that of the mouse and other mammals, one may expect that a
few dozen germ cells, originating from the proximal region of the embryonic
86
ectoderm, start their journey inside the embryo along with the invaginating
hindgut. A recent study in mice showed that expression of Bmp4 (bone
morphogenetic protein 4 gene) in the trophectoderm layer which is in closest
contact with the epiblast is responsible for the differentiation of both the
primordial germ cells and the allantois (Lawson et al., 1999). If a similar mech-
anism operates in the horse, BMP4 would presumably be produced by the
polar trophoblast cells which overlie the inner cell mass before the former are
lost, i.e. before day 12 (Enders et al., 1993). Due to ongoing proliferation, a
significant number of germ cells reach the genital ridge, which consists of a
thin layer of mesenchymal cells located between the coelomic epithelium and
the mesonephros. Two genes, Sf1 and Wt1, are particularly important in devel-
opment of the murine genital ridge (McLaren, 1998). Eventually, four different
cell lines comprise the genital ridge: primordial germ cells, somatic steroido-
genic cells, supporting cells and connective tissue. The fate of each lineage
depends on the sexual determination of the embryo in which they develop,
and their structure, function and pattern of genetic activity is quite different in
testes and ovaries.
A study using alkaline phosphatase staining, to investigate the distribution
of primordial germ cells in early equine embryos (Curran et al., 1997), detected
only one or two positive cells in embryos at day 20 and about 3267–2424 cells
at days 28–30. However, unlike other species, a large number of these positive
cells (72% at day 28) were found in the vascular system and other organs with
only 28% in the genital ridges, suggesting possible germ cell migration via the
vascular system (Curran et al., 1997).
It was known long ago that sex determination in mammals depended on
the presence or absence of the Y chromosome. Embryos without a Y chromo-
some develop as females and those with a Y chromosome develop as males.
The breakthrough in molecular understanding of sex determination and differ-
entiation in the mouse and human (Goodfellow and Lovell-Badge, 1993)
paved the way for other mammals including the horse. In humans and mice
and probably other mammals, gonadal sexual differentiation starts relatively
late in embryonic development, and morphological differences in XY embryos
appear prior to XX embryos. In the horse, sexual differentiation of the gonads
occurs by about day 40 (Table 13.1). It is likely that this differentiation starts in
males several days earlier than in females (Merchant-Larios, 1979).
Entry of the oocytes into meiotic prophase occurs later during gestation.
The first meiotic prophase begins in equine fetal ovary cells at days 60–70.
Later, at 150–200 days, oocytes in early meiotic stages fill the ovarian cortex
(Deanesly, 1977). The great majority of the oocytes first involved in meiotic
divisions disappear, and only a small number of them later develop into
primordial follicles. Spermatocytes enter meiosis during postnatal life and it
was found recently that the murine male genital ridge at about 12 days
post-coitum produces a factor that inhibits entry of germ cells into meiosis
(McLaren and Southee, 1997).
Testicular development is a key element in establishing mammalian sex.
The chromosomal constitution determines the migration of cells into gonads
87
and the final differentiation into a testis or an ovary (Hunter, 1995). Testicular
development in mammals is triggered by a gene on the Y chromosome encod-
ing the testis-determining factor (TDF), or sex determining region of the Y
chromosome (SRY). In genetic males, this factor induces differentiation of
Sertoli cells (reviewed by McLaren, 1991) and secretion of anti-Müllerian
hormone (AMH). AMH, which belongs to the TGF-β family, causes regression
of Müllerian ducts, promotes development of Wolffian ducts and the differenti-
ation of Leydig cells which secrete the male steroid hormone, testosterone
(Behringer, 1995). Testosterone binds to androgen receptors, which in turn act
as transcription factors. Further details about AMH and its activity in bovine
development are presented elsewhere (Cate and Wilson, 1993).
Differentiation of somatic cells into steroidogenic cells takes place in
horse embryos very early during development. The seminiferous cords of the
developing testis are completely segregated from the steroidogenic tissue by
basal lamina, while in the medulla of the ovary, steroidogenic cells differenti-
ate inside the epithelial cords which contain germ cells (Merchant-Larios, 1979;
Knospe and Budras, 1992; Knospe, 1998). Details about further sexual differ-
entiation in equids are not available, but in bovine fetuses, regression of the
Müllerian ducts occurs in males between 50 and 80 days of development
(Vigier et al., 1984). A whole chain of developmental events follows, and the
phenotype typical for males arises. In females, Müllerian ducts develop, no
Leydig cells form in the gonad, no testosterone is produced and gonad
development steadily moves towards the female phenotype. The female devel-
opmental programme is therefore the ‘default’, while the male programme
requires switching on of the SRY gene followed by a cascade of activation of
autosomal genes.
From around day 80 of gestation, the gonads of both male and female
equine fetuses undergo extraordinary proliferation and growth such that, by
day 250 of gestation, they may weigh as much as 50 g each and are usually
much larger than the maternal ovaries (Hay and Allen, 1975). Proliferation of
the interstial cells causes this enlargement and these cells secrete large quanti-
ties of C-19 steroids which are aromatized in the placenta to both the phenolic
oestrogens, oestrone and oestradiol and the unique ring B unsaturated oestro-
gens, equilin and equilinin. Beyond day 280, the gonads begin to regress so
that at birth they are of normal size and histological appearance. Bilateral fetal
gonadectomy results in retarded growth of the fetus and abnormal parturition,
thereby suggesting that the large quantities of oestrogens produced by the
equine fetoplacental unit are involved in fetal growth and in preparing
the uterus for parturition (Pashen and Allen, 1979).
SRY gene and sexual differentiation
The testis-determining role of the SRY gene in mammals is widely accepted

after the experiments performed in the early 1990s (reviewed by Goodfellow
and Lovell-Badge, 1993). Available data also suggest that the cell-autonomous
88
activity of the murine Sry gene in Sertoli cell precursors results in differentia-
tion of Sertoli cells (Burgoyne et al., 1988). Polymerase chain reaction (PCR)
products of the horse HMG box, the only region of SRY that is conserved
between species (Lovell-Badge, 1993), have been sequenced (Meyers-Wallen
et al., 1997) and are available from GenBank. The complete coding sequence
of cDNA (1420 bp) for the equine SRY gene has been determined just recently.
Contrary to the situation in mice, the equine linear RNA transcript in testicular
tissue was expressed just after puberty (Hasegawa et al., 1999).
After cloning of the SRY gene and the demonstration that it was a tran-
scription factor (Ramkissoon and Goodfellow, 1996; Greenfield, 1998), several
autosomal genes acting downstream of SRY were shown to be involved in the
mammalian sex differentiation pathway. This set of genes includes the SRY-
related high-mobility group box (SOX) autosomal gene family, which display
properties of both classical transcription factors and architectural components
of chromatin (Pevny and Lovell-Badge, 1997). Sox9 has an essential function in
sex determination in mammals and is critical for Sertoli cell differentiation
(Morais da Silva et al., 1996). The human DAX-1 gene and its mouse homo-
logue are located on the X chromosome and encode an unusual member of
the nuclear hormone receptor superfamily. Mutations in this gene cause
adrenal hypoplasia (Greenfield, 1998). The autosomal SF-1 gene produces
another nuclear receptor, steroidogenic factor 1. Mutations in this gene may
cause gonadal and adrenal agenesis and other disorders. These genes act at
the same time (Greenfield, 1998).
Cycle of the X chromosome
As proposed by Lyon (1961), it is now accepted that one of the X chromo-

somes in mammalian females undergoes inactivation during early embryonic
development. Numerous investigations have shed light on different aspects of
X chromosome behaviour, including preferential inactivation of the paternal X
chromosome in trophoblast, random inactivation in the ICM, and molecular
mechanisms of inactivation (Goto and Monk, 1998).
These data appear to be fully applicable to the cycle of the X chromosome
in the horse. Inactivation of the X chromosome in equine XX embryos begins
gradually in trophoblastic cells around day 7.5 and in the embryonic disc
around day 11.5. Cells with an inactive X chromosome predominate in the
trophoblast by day 10.5 and in the disc by day 12.5 (Romagnano et al., 1987).
In post-meiotic oocytes, the X chromosomes should become active again, as
observed in mice and humans. The paternal X chromosome enters the zygote
inactive but, soon after fertilization, the paternal X chromosome reactivates in
XX embryos and both X chromosomes are active until trophoblast differentia-
tion. At this time, preferential inactivation of the paternal X chromosome takes
place in trophoblast cells (days 7.5–10.5). In the ICM (embryonic disc), both X
chromosomes are active for some time, but later one of them undergoes
random inactivation (days 11.5–12.5) (Serov et al., 1978; Romagnano et al.,
89
1987). The promoter region of a key gene involved in X inactivation (XIST)

was cloned recently from a horse genomic library and used, along with the
equivalent human, mouse and rabbit sequences, to identify the minimal
promoter region of the XIST gene (Hendrich et al., 1997).
Sterility associated with a lack of one X chromosome (XO karyotype)
observed in the mare (Bowling et al., 1987; Buoen et al., 1993) appears similar
to the human XO disorder, but different from the mouse condition. It could be
an additional indication of minor variations in the pattern of X chromosome
inactivation in divergent mammalian species (Goto and Monk, 1998). Different
forms of mosaicism (chimaerism) for X chromosomes described in horses
cause abnormalities of sexual differentiation (Power and Leadon, 1990) and
infertility (Gill et al., 1988).
Sex reversals in the horse
As indicated above, the SRY locus plays a crucial role in sex differentiation and,
in the normal situation, only embryos carrying an Y chromosome possess SRY.
However, SRY can be non-functional or transferred from the Y chromosome
to the X chromosome by a rare recombination event. These events can cause
complete sex reversal whereby XY individuals become females and XX
individuals become males (Cattanach et al., 1982).
The XY sex reversal syndrome has been described in the domestic horse.
In several cases, the progeny of stallions showed significant deviation from the
expected sex ratio, as well as an increased level of female infertility (Kent
et al., 1986). Also, a karyotype indistinguishable by G- or C-banding from that
of a male horse (64,XY) was common in mares with development defects
(Bowling et al., 1987; see also Chapter 9, pp. 188–191). Molecular analysis of
an XY mare showed that at least the DNA-binding domain of the SRY gene was
deleted from the Y chromosome (Pailhoux et al., 1995). An investigation of 38
mares with the XY sex reversal syndrome identified four classes with different
degrees of abnormality. These include: (i) normal females, some of which
were fertile; (ii) females with gonadal dysgenesis and normal Müllerian duct
development; (iii) intersex with gonadal dysgenesis, enlarged clitoris and
abnormal Müllerian duct development; and (iv) virilized intersex with high lev-
els of testosterone. Usually, the two former classes were H-Y negative whereas
the two later classes were H-Y positive (Kent et al., 1988a, b).
The opposite situation was also described. A bilateral cryptorchid stallion
with mild development of mammary glands was identified by karyotyping as
an XX male. Underdeveloped accessory sex organs and hypoplastic, inguinally
located testes that were deficient in spermatogonia were found in this stallion
(Constant et al., 1994). The cause of this rare syndrome is unknown but
it could be related to SRY abnormalities. However, SRY-negative XX true
hermaphroditism in a horse also appears possible (Meyers-Wallen et al., 1997).
One described Pasa Fino horse had ovotestes, no Müllerian or Wolffian duct
derivatives, a blind-ending vagina and an enlarged clitoris. It was diagnosed
90
SRY-negative by PCR analysis, suggesting that the masculinization effects

during development were due to expression of genes downstream of SRY in
the male developmental pathway.
Development of Interspecies Hybrids

Description of interspecies hybrids
Equids display a remarkable ability to interbreed between the member

species of the genus, and the mule (female horse ×male donkey), as well as
being produced for centuries as an efficient work animal, has also provided
many insights into the genetics and evolution of speciation (Short, 1975).
Mules are much more common than the reciprocal cross, the hinny (female
donkey ×male horse) because, for some unknown reason, the latter mating is
not as fertile as the former. Both mules and hinnies, although infertile, are
stronger and live longer than either parental species, thus displaying ‘hybrid
vigour’. This was recognized in at least 1000 years BC, which led to their exten-
sive use in war during succeeding generations, particularly as pack animals.
Mules are still used today in many parts of the world as work animals.
Not only is hybridization possible between all the equine species (Gray,
1972), the transfer of embryos between the different species is also frequently
successful (Allen and Short, 1997). An underlying reason for this remarkable
ability of female equids to carry inter- and extra-specific conceptuses of very
different genetic constitution may relate to the non-invasive nature of the
equine placenta and the retention of six layers of tissue separating the mater-
nal and fetal blood supplies (Samuel et al., 1976). It is also likely to be influ-
enced by the immunological responses of the mother to the development of
the endometrial cups and to the absence of paternally derived MHC antigens
on the surface of the non-invasive trophoblast cells of the allantochorion
(Allen et al., 1984; Donaldson et al., 1990).
Contribution of equine hybrids to developmental genetics
In addition to being useful as work animals, equine hybrids have made a

significant contribution to scientific thought, including ideas on developmental
genetics (Short, 1975). The earliest example was Aristotle’s realization that,
based on the physical appearance of mules, there was a more or less equal
contribution from both parents to the phenotype of the offspring, rather than
the female playing a purely passive role. However, it was not until the 17th
century that sperm and eggs were discovered and the mechanism of sexual
reproduction via fusion of the gametes was realized (Short, 1975). Equine
hybrids have also contributed to thinking on gestation length and the control
of parturition. Gestation lasts about 11 months in the horse and about 13
months in the donkey. Mule and hinny fetuses, on the other hand, are both
91
carried for around 12 months (Allen et al., 1993), thereby indicating that the
fetus, not the mother, is the major determinant of gestation length.
Other areas in which equine hybrids have proved useful are those
involving the importance of chromosome pairing during meiosis and the
influence of viable germ cells on gonadal development. The difference in
the number and structure of chromosomes present in each parent underlies
the block to meiosis which occurs in hybrid offspring (Chandley et al., 1975).
However, occasional oocytes have been observed in sections of mule fetal
ovaries (Taylor and Short, 1975).
The importance of viable germ cells for normal gonadal differentiation has
been studied in male equine hybrids. The testes of three male hinnies were
examined by light and transmission electron microscopy to observe the
development of germ cells and to verify morphological modifications due
to the hybridization (Landime Alvarenga and Bortolozzi, 1994). The hinny
seminiferous epithelium contained Sertoli cells and spermatogonia with
normal features, but the primary spermatocytes appeared abnormal and other
cells in the spermatogenic sequence were not present. Most of the alterations
began to occur in the primary spermatocytes, which showed nuclear vacuol-
ization and deposits of amorphous material between the carioteca and the
nuclear lamina to form vesicles or exaggerated chromatin condensation which
resulted in pyknosis. Vacuolization and organelle destruction was also
observed in the cytoplasm. The arrest of meiosis due to lack of chromosome
homologies leads to germinal cell degeneration and, therefore, the arrest of
spermatogenesis. This, in turn, causes a profound alteration in the morphology
of the seminiferous epithelium.
Equine hybrids have also been used to study the phenomenon of X
chromosome inactivation. The Lyon hypothesis (Lyon, 1961, 1970) stated that
only one of the two X chromosomes was active in the somatic cells of female
mammals and that inactivation was a random event which took place early in
embryonic life. Female mules and hinnies provided the ideal test situation
and Hamerton et al. (1971) used the expression of species-specific glucose-6-
phosphate dehydrogenase (G6PD) to show that, in any given cell, only one of
the two X chromosomes is functional. This was later examined in more detail
using starch gel electrophoresis of erythrocyte G6PD recovered from 42 female
and 32 male mules, 35 donkeys and ten horses (Serov et al., 1978). The
quantitative expression of the parental alleles at the GPD locus varied greatly
in female mules, from hemizygous expression of the maternal allele to that of
the paternal, thereby confirming random inactivation in females mules. No
selective advantage of a cell population with a maternally (or paternally)
derived active X chromosome was found.
Equine hybrids will continue to play an important role in research on
developmental genetics in the future. For example, the role of gametic
imprinting in placental and fetal development should be very fruitful, and
progress in this area is bound to be stimulated as more and more equine
developmental genes and their products are identified.
92
A3759:
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NSW 2351, Australia; 2TBA Equine Fertility Unit, Woodditton Road,

344 A. Ruvinsky and F. Stewart

Developmental Stages of the Horse Embryo

Developmental Genetics 345

Stage of development Days after ovulation Cells/stage/crlb

Vesicular (ovum) period 0–15

346 A. Ruvinsky and F. Stewart

Developmental Genetics 347

348 A. Ruvinsky and F. Stewart

These structures secrete large amounts of equine chorionic gonadotrophin

Genetic Control of Pre-implantation Development

There is considerable evidence in lower species that very early development is

Developmental Genetics 349

Electron microscopy studies suggest that activation of the horse embryonic

350 A. Ruvinsky and F. Stewart

pre-implantation development in the mouse embryo. A similar picture is

Embryonic gene expression

Blastocyst formation creates two different cell lineages: non-polarized inner

Developmental Genetics 351

352 A. Ruvinsky and F. Stewart

Developmental Genetics 353

Trophoblast gene expression

Differentiation of trophoblast cells, the first differentiation event during

354 A. Ruvinsky and F. Stewart

growth of equine trophoblast cells. A separate gene, Hed, encodes a related

Developmental Genetics 355

prevents maternal immune rejection of the fetal–placental unit. The expression

The fundamental assumption of Mendelian genetics is that the behaviour of

356 A. Ruvinsky and F. Stewart

Developmental Genetics 357

A study of eCG concentration in the serum of pregnant mares and

358 A. Ruvinsky and F. Stewart

Maternal Recognition of Pregnancy and Placentation

Development of the placenta

Placentation in the mare is a prolonged process, involving a transient yolk sac

Developmental Genetics 359

has disintegrated, and the gradual development of the chorioallantoic placenta

360 A. Ruvinsky and F. Stewart

Genes Involved in Control of Morphogenesis

Gastrulation in the horse starts on days 13–14 of development (Ginther, 1992).

Developmental Genetics 361

development, including notochord formation, is discussed below. The next

Hox genes and development of axial identity

362 A. Ruvinsky and F. Stewart

a striking case of evolutionary conservation. The Hox gene family determines a

Organogenesis: T-box and Pax genes

Developmental Genetics 363

364 A. Ruvinsky and F. Stewart

evolution to play a role in the differential specification of fore- (pectoral)

Information on development of muscle tissue in the horse is also important

Developmental Genetics 365

Developmental effects of coat colour mutations

Classical coat colour genetics in mammals has acquired developmental and

366 A. Ruvinsky and F. Stewart

intestinal aganglionosis (Hultgren, 1982) which results in a poorly developed

The earliest stages of gonadal development in mammals occur at a similar

Developmental Genetics 367

368 A. Ruvinsky and F. Stewart

SRY gene and sexual differentiation

The testis-determining role of the SRY gene in mammals is widely accepted

Developmental Genetics 369

Cycle of the X chromosome