&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-96/079
July 1996
Prehatching
Development of the
Fathead Minnow
Pimephales Promelas
Rafinesque
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EPA/600/R-96/079
July 1996
PREHATCHING DEVELOPMENT OF THE FATHEAD MINNOW
PIMEPHALES PROMELAS RAFINESQUE
By
E. W. Devlin,1 J. D. Brammer,2 R. L. Puyear,2 and J. M. McKim3
'Biology Department, Hampden-Sydney College, Hampden-Sydney VA
2Zoology Department, North Dakota State University, Fargo, ND 58105
3USEPA, Mid-Continent Ecology Division, Duluth, MN 55804
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
Printed on Recycled Paper
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NOTICE
This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names or commercial products does not constitute endorse-
ment or recommendation for use.
n
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ABSTRACT
The fathead minnow, Pimephales promelas Raf., presents a classical model of teleostean embryogen-
esis. Its prehatching development has been divided into 32 stages, each representing an easily observed
interval in the developmental continuum. Embryos were examined live and histologically under con-
trolled laboratory conditions. Fertilization, early cleavage, epiboly, and organogenesis are very similar to
that of other cyprinids except for the timing of the appearance of specific structures. Hatching was found
to occur in approximately 120 hours post-fertilization at 25°C. Rapid embryonic development, coupled
with a short generation time of 3-6 months under laboratory conditions, make it a useful native North
American species for studies in experimental embryology.
in
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TABLE OF CONTENTS
Notice ii
Abstract iii
Table of Contents iv
List of Tables v
List of Plates vi
Glossary vii
Acknowledgements viii
INTRODUCTION 1
MATERIALS AND METHODS 3
RESULTS AND DISCUSSION 3
Developmental Stages:
1. Unfertilized ovum 8
2. Recently fertilized ovum 9
3. 1-celled blastodisc 10
4. 2-celled blastodisc 11
5. 4-celled blastodisc 12
6. 8-celled blastodisc 13
7. 16-celled blastodisc 14
8. 32-celled blastodisc 15
9. Late cleavage 16
10. High blastula 17
11. Flat blastula 18
12. Early gastrula 19
13. One-quarter epiboly 20
14. One-half epiboly 21
15. Three-quarter epiboly 22
16. Closure of germ ring 23
17. Neurula stage, 4-5 somite pairs 24
18. Optic vesicles. 9-10 somite pairs 25
19. Neuromeres, 14 somite pairs 27
20. Otic vesicle, 16 somite pairs 28
21. Tailbud stage. 18-20 somite pairs 29
22. First movements, lens formation 30
23. Heartbeat without circulation 31
24. Onset of circulation 32
25. Retinal pigmentation 34
26. Blood in intersegmental arteries 36
27. Horizontal duct in otic capsule 38
28. Blood flow in pectoral fins 40
29. Formation of yellow bile 42
30. Dorsally pigmented swim bladder 43
31. Large operculum, limited hatching 45
32. Gill anlagcn. hatching 46
REFERENCES 47
iv
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LIST OF TABLES
Number Page
1. Selected species whose embryonic development has been described 2
2. Characterization of embryonic stages of Pimephales promelas at 25°C 4
3. A preliminary time sequence of the larval Stages of Pimephales promelas from hatching
through the juvenile phase of development at 25°C 5
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LIST OF PLATES
Number Page
1. Unfertilized ovum 8
2. Recently fertilized ovum 9
3. 1-celled blastodisc 10
4. 2-celled blastodisc 11
5. 4-celled blastodisc 12
6. 8-celled blastodisc 13
7. 16-celled blastodisc 14
8. 32-celled blastodisc 15
9. Late cleavage 16
10. Highblastula 17
11. Flatblastula 18
12. Early gastrula 19
13. One-quarter epiboly 20
14. One-half epiboly 21
15. Three-quarter epiboly 22
16. Closure of germ ring 23
17. Neurula stage, 4-5 somite pairs 24
18. Optic vesicles, 9-10 somite pairs 25
19. Neuromeres, 14 somite pairs 27
20. Otic vesicle, 16 somite pairs 28
21. Tailbud stage, 18-20 somite pairs 29
22. First movements, lens formation 30
23. Heartbeat without circulation 31
24. Onset of circulation 32
25. Retinal pigmentation 34
26. Blood in intersegmental arteries 36
27. Horizontal duct in otic capsule 38
28. Blood flow in pectoral fins 40
29. Formation of yellow bile 42
30. Dorsally pigmented swim bladder 43
31. Large operculum, limited hatching 45
32. Gill anlagen, hatching 46
VI
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GLOSSARY
3v Third ventricle Li
4v Fourth ventricle LiS
A Anterior LJ
AA Aortic arches LP
B Blastomere LS
BD Bile duct M
Bl Blastoderm Me
C Chorion Me
Ca Caudal MF
CA Caudal artery
cc Common cardinals MFF
CF Choroid fissure Mt
ChP Choroid plexus Mv
CK Caudal knob N
Cl Cloaca Nc
GIF Cleavage furrow NC
Co Coelomic cavity NCr
CP1 Cleavage planes Nk
CV Caudal vein Nm
Cy Cytoplasm Nmt
D Diencephalon Nu
DT Dorsal tube O
EE Epidermal ectoderm OA
EmS Embryonic shield OC
Ep Epiphysis OG
ES Epidermal stratum O1P
Fg Foregut ON
G Gut ONL
GA Gill arches Op
GB Gallbladder Otc
GL Ganglion cell layer OtP
GR Germ ring OtV
H Heart OV
HA Heart anlage P
HE Head ectoderm PC
HR Head region PCo
HV Heart ventricle PD
I Infundibulum PF
INL Inner nuclear layer Ph
IPL Internal plexiform layer PN
KV Kupffer's vesicle Pr
L Lens PS
LD Line drawing Pt
LDi Liver diverticulum PT
Liver PvS
Liver sinusoids R
Lower jaw RC
Lens placode S
Live specimen HS
Micropyle SA
Melanocytes SB
Mesencephalon Sc
Mesen-metencephalon SC
fissure SEM
Median fin fold
Mitotic figures Sg
Microvilli SM
Notochord St
Neurocoele StC
Nerve Cord T
Neural crest cells TB
Neural keel TL
Neuromeres TP
Neuromast V
Nucleus WM
Otoliths Y
Optic anlage YIP
Optic cup YP
Olfactory groove
Olfactory placode
Optic Nerve
Outer nuclear layer
Operculum
Otic capsule
Otic placode
Otic vesicle
Optic vesicle
Periblast
Periblast corona
Pericardial coelom
Pronephric ducts
Pectoral fin
Pharynx
Periblast nuclei
Prosencephalon
Paraffin section
Proctodeum
Pronephric tubule
Perivitelline space
Rhombencephalon
Radial cartilage
Somite
Hyaline sheath
Somite anlage
Swim bladder
Sclerotic coat
Spinal cord
Scanning electron
microscope
Segmentation cavity
Segmental mesoderm
Stomach
Striated cartilage
Telencephalon
Tailbud
Tectal lobe
Thickened periblast
Unlined vesicle
White matter
Yolk (mass)
Yolk plug
Yolk particles
vn
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ACKNOWLEDGEMENTS
We wish to thank Dr. Conrad Firling, University of Minnesota, Duluth and Dr. Rodney Johnson,
Environmental Research Laboratory, Duluth for their review and comments on the Manuscript. Finally,
this research was partially funded by Air Force Research Grant AFOSR-78-3709, and National Institutes
of Health Environmental Pathology Training Grant 5T32ES07032.
vin
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INTRODUCTION
The use of fish embryos as models to study the action of toxic substances has been going on since the
early 1900's with the work of Stockard (1910), McClendon (1912), and others. These workers recog-
nized their utility as model organisms, offering many advantages over mammalian systems: the embryo
is contained within a chorion which is often clear allowing observation of embryogenesis without pertur-
bation of the embryo; teleost embryos are often easily cultured in the lab year-round; many species offer
large numbers of eggs from one female (minimizing much of the variability when using gametes from
many individuals), depending on the species chosen, a large range of developmental rates are available
(Table 1).
Different teleostean species exhibit a range of developmental complexity at hatching. This is seen in
the developmental stage of the musculature, integument and skeletal or the extent of yolk utilization. For
example, in the slower developing salmonids the newly hatched larvae possess a large yolk sac which
results in limited mobility. At hatching the fathead minnow, Pimephales promelas, is well developed
possessing a streamlined yolk sac, partial body pigmentation, an open mouth with movable jaws, well
developed musculature, eyes and neuromasts system (as evidenced by attempts to catch the newly
hatched larvae) and is able to feed shortly following hatching. It should be noted that this post-hatch
period between complete utilization of yolk stores and exogenous feeding represents a time of metabolic
stress in which the young larvae are especially vulnerable to stress (Devlin, 1982). The present study has
concentrated on development up to hatching, further work on the later larval stages would also be useful.
The fathead minnow is a small hardy cyprinid that is an important forage fish (Lord, 1927). Its life
cycle has been described by Markaus (1934), Bullough (1939), Andrews (1970), and Andrews and
Flickinger (1974). The relatively simple procedure for obtaining fathead minnow eggs, as well as its
short generation time of 3 to 6 months under controlled conditions, has made it useful in toxicological
studies (Benoit and Carlson, 1977; McKim, 1977; Till, 1977). Yet in spite of its ecological significance
and widespread use in laboratory studies, descriptions of its early development are incomplete. Partial
descriptions of its embryonic development have been reported (Niazi, 1963; Manner and DeWese, 1974;
Wabuke-Bunoti, 1980).
These descriptions are useful, but do not allow investigators to accurately assign a stage of develop-
ment, nor do they provide a complete detailed description of normal development.
Many accounts of teleost embryogenesis are available (Table 1). In reviewing this literature, the
timing of appearance of embryonic structures is seen to differ among teleosts. Indeed, although general-
ized patterns of development exist, there is enough variation to make any standard staging sequence
beyond the limits of the suborder unreliable (Long and Ballard, 1976). The present study provides a
detailed embryonic staging sequence, which will allow a more widespread use of the fathead minnow as
a model organism in laboratory studies.
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Table 1. Selected species whose embryonic development has been described.
Number
Age at of
Species Hatch (hrs) Temp (°C) Stages Author
Abudefduf saxatilis
Adinia xenica
Austrofundulus myersi
Barbus barbus
Brachydanio rerio
Carassius auratus
Catostomus commersoni
Coregonus clupeaformis
Erimyzon sucetta
Esox masquinongy
Fundulus heteroclitus
Gasterosteus aculeatus
Heteropneustes fossilis
Ictalurus punctatus
Macropodus opercularis
Micromesistius poutassou
Micropterus dolomieui
Myoxocephalus aenaeus
Notropis bifrenatus
Oncorhynchus kisutch
Oncorhynchus keta
Oncorhynchus nerka
Oryzias latipes
Perca flavescens
Pimephales promelas
Polyodon spathula
Pomatomus saltatrix
Pseudopleuronectes americanus
Salnio gairdneri
Salnw solar
Serranus atrarius
Tilapia galilae
Trichogaster trichopterus
158*
239*
1248
194
96
100
492*
3120
106*
336
228
140
7*
—
36
20
44*
1008*
57
960
—
1170
264
848
—
240
47*
360
600
4444*
75
48
23*
24
27
25
16
26
21
10
10
21
13
20
17
28
26
27
8
23
6
26
10
8
10
25
10
23
14
20
2
10
1
16
24
26
19
33
46
7
25
25
22
803
21
—
34
—
—
19
—
4
16
—
15
—
16
30
36
8
12
36
—
—
23
34
—
4
—
Shaw, 1955
Koenig & Livingston, 1 976
Wourms, 1972
Penaz, 1973
Hisaoka & Battle, 1958
Kajishima, 1960
Long & Ballard, 1976
Price, 1934; Price, 1935
Shaklee et al., 1974
Galat, 1973
Armstrong & Child, 1965
Vrat, 1949
Thakuretal., 1974
Saksena et al., 1961
Allen & Mulkay, 1960
Coombs &Hiby, 1979
Wallace, 1972
Lund & March, 1975
Harrington, 1947
Zeitoun, 1974
Mahon&Hoar, 1956
Velsen, 1980
Kirchen& West, 1976
Mansueti, 1964
Manner & Dewese, 1974
Ballard & Needham, 1964
Deueletal., 1966
Breder, 1922
Ballard 1973
Battle, 1944
Wilson, 1891
El Zarka & Ezzat, 1972
Hodges & Behre, 1953
* Midrange of the reported range of values
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METHODS AND MATERIALS
Fathead minnows were obtained from stock cultures at the U.S. Environmental Research Laboratory,
Duluth, MN (Denny, 1987). Breeding facilities in our laboratory consisted of 28 10-liter tanks, each
containing a U-shaped tile, one mature male, and two mature females. All tanks were fitted with
standpipes and individually supplied with air and water. City water (pH 8.3, hardness 80 mg/1 CaCOS)
passed through activated carbon was used in all tanks. Water temperature was maintained at 24°C+0.5.
The whole culture system was enclosed in a light-tight insulated room, with a photoperiod of 16 hr light
(100 lux at the water surface) and 8 hr dark. Mature fish were fed frozen brine shrimp and Glenco No. 2
enriched trout food twice daily. Larval fish were fed brine shrimp twice daily.
In order to observe events at fertilization, mature ova were obtained by removal of fully developed
ovaries from gravid females and added to sperm suspensions obtained from mature minced testis. To
observe later stages, embryos were removed from the underside of the U-shaped tiles in the breeding
tanks, placed in aerated 2-liter tanks maintained at 25°C+0.2, and maintained under the same photope-
riod (25 lux at the water surface) as the adults. At predetermined intervals, 5 to 10 embryos were gently
removed from the tiles for observation.
Embryos for light microscopy were dissected out of their chorion, photographed with a compound
microscope, fixed in formalin-acetic acid-alcohol (FAA) (Humason, 1979), embedded in paraffin,
serially sectioned at 6 (land 10 p, and stained with Harris hematoxylin and eosin. Sections were photo-
graphed with a compound microscope. Measurements of average length were determined with an ocular
micrometer calibratedin hundredths of a millimeter from a sample of 15 embryos.
Embryos for scanning electron microscopy were fixed in 4% phosphate-buffered glutaraldehyde (pH
7.4) overnight, rinsed three times in buffer, then postfixed in 2% OsO4 for 3-5 hr. Fixations were carried
out at 4°C. Following osmium fixation, embryos were again rinsed in buffer, dehydrated with ethanol at
room temperature, and immediately critically point dried with liquid CO2 in a Samdri PVT-3. Embryos
were mounted on stubs and coated with gold in a Hummer II sputter coater. A JOEL JSM-35 scanning
electron microscope was used to examine specimens.
RESULTS AND DISCUSSION
This study describes the prehatching development of the fathead minnow under controlled laboratory
conditions in which the development is divided into 32 stages (Table 2). In addition, preliminary
posthatch descriptions of 9 larval developmental stages were made on this minnow at 25°C (Table 3).
The following detailed fathead minnow prehatching development sequence is described in 32 indi-
vidual stage descriptions with accompanying photographic plates:
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Table 2. Characterization of embryonic stages of Pimephales promelas at 25°C.
Dev. Stage Age (hours) Length (mm) Characteristics of the Embryo Stage
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0.00
0.16
0.66
1.00
1.33
1.58
2.25
2.83
3.50
4.50
5.25
6.00
7.33
9.50
12.00
15.00
16.50
17.75
20.00
22.00
24.00
26.00
30.00 2.19
35.33 2.83
40.00 3.35
50.00 3.56
60.00 3.89
74.00 4.53
85.00 4.90
95.00 5.10
105.00 5.18
120.00 5.14
Unfertilized ovum
Recently fertilized ovum
1 -celled blastodisc
2-celled blastodisc
4-celled blastodisc
8-celled blastodisc
1 6-celled blastodisc
32-celled blastodisc
Late cleavage
High blastula
Flat blastula
Early gastrula
One -quarter epiboly
One-half epiboly
Three-quarter epiboly
Closure of the germ ring
Neurula stage, 4-5 somite pairs
Optic vesicles, 9-10 somite pairs
Neuromeres, 1 4 somite pairs
Otic vesicle, 16 somite pairs
Tailbud stage, 18-20 somite pairs
First movements, lens formation
Heartbeat without circulation
Onset of circulation
Retinal pigmentation
Blood in intersegmental arteries
Horizontal duct in otic capsule
Blood flow in pectoral fins
Formation of yellow bile
Dorsally pigmented swim bladder
Large operculum, limited hatching
Gill anlagen, hatching
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Table 3. A preliminary time sequence of the larval stages* of Pimephales promelas from hatching
through thejuvenile phase of development at a mean water temperature of 25°C.
Dev. Stage Age in Hours (post-hatch) Characteristics of Larval Stage
1 12.0 protolarval phase
(median fin fold)
2 24.0 posterior air bladder filled
3 32.0 yolk-sac disappears
4 56.5 exogenous feeding
98.5 mesolarval phase
(partial fin ray development)
223.0 anterior air bladder filled
273.0 metalarval phase
(pelvic fin buds)
299.0 caudal fin, symmetrical
9 443.0 juvenile phase
(fin development complete)
* Approximately 50 larval fish were used to establish these 9 posthatch larval stages.
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FATHEAD MINNOW PREHATCHING DEVELOPMENT
PLATES 1 - 32
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Stage 1: Unfertilized ovum. Plate 1
The mature unfertilized ovum is spherical and surrounded by a closely appressed chorion (C). The
distinctive micropyle (M) is funnel-shaped with 7 ridges, and protrudes into the yolk mass which con-
sists of yolk platelets mixed with cytoplasm. Cortical granules in the cortical cytoplasmic layer are
difficult to observe, but lie on the surface of the whitish, translucent yolk mass. The perivitelline space
has not yet formed as seen in Figure A (80x, LS). The opaque yolk mass and cytoplasm of the ovum is
represented by small dots in Figure B (80x, LD).
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Stage 2: Recently fertilized ovum.
Plate 2
Following fertilization, the cortical granules break down, starting in the region just under the micro-
pyle. This results in the separation of the chorion from the cytoplasmic layer, with the subsequent forma-
tion of the perivitelline space. Large yolk platelets appear to flow together, and the yolk mass becomes
clear and remains clear throughout subsequent development. In living embryos, cytoplasm appears to
migrate through and around the yolk mass to the animal pole where a low, broad cytoplasmic cap, the
blastodisk is forming, establishing the polarity of the embryo.
The chorion has not yet water-hardened and is still soft and fragile, making it difficult to collect
embryos without damaging them. The thin plasma membrane which completely surrounds the embryo is
covered with microvilli. In later stages, these microvilli will only be found on the blastomeres, but not
on the plasma membrane surrounding the yolk mass. Figure A shows the early cytoplasmic cap (Cy)
visible above the yolk mass (Y) (80x, SEM). Figure B is a dechorionated live embryo which shows the
cytoplasmic cap as an elevated clear region of the peripheral cytoplasm. The yolk has started to clear,
although aggregates of large yolk platelets are still present. Note this embryo and all subsequent micro-
graphs of live embryos have been dechorionated (80x, LS). In addition to the cytoplasmic cap and yolk
mass, Figure C shows the surrounding chorion (C) with its micropyle (M) and the perivitelline space
(PvS) that has formed (80x, LD).
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lv*
^*£fe
x'j
Stage 3: One-celled blastodisc.
Plate 3
The one-celled blastodisc consists of a large cytoplasmic cap (Cy) which bulges above the yolk mass
(Y). Small yolk particles (YP) are found in the granular cytoplasm of the blastodisc. The blastodisc is in
close contact with the yolk and is continuous with a thin cytoplasmic layer covering the yolk. In Figure
A the cytoplasmic cap is visible as an elevated cap on the yolk mass. The smooth transition of the cyto-
plasmic cap into the yolk mass seen in this figure is not present in living embryos and is thought to be a
fixation artifact (80x, SEM). In Figure B the cytoplasmic cap appears as a clear elevated mass of cyto-
plasm on the yolk mass (80x, SEM). Figure C shows small yolk particles (YP) visible in the cytoplasm
of the cytoplasmic cap (lOOx, PS). Figure D shows the chorion (C), perivitelline space (PvS), cytoplas-
mic cap, as well as the residual cytoplasm (Cy) still present in the yolk mass. The solid line shown in
this and subsequent drawings represents the ventral border of the early blastomeres. This border is in
fact a transition zone rather than an abrupt line (80x, LD).
10
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CIF
Stage 4: Two-celled blastodisc.
Plate 4
The first cleavage plane appears initially as a slight depression in the blastodisc and then deepens into
a vertical furrow. The first cleavage takes about 7 minutes from start to finish at 25°C. The division is
meroblastic and meridional or vertical, resulting in two equal-sized blastomeres (B) which are elevated
above the yolk mass (Y). The cleavage furrow (GIF) separating the two blastomeres is visible in Figure
A (66x, SEM), Figure B (80x, LS) and Figure C (80x, LD); chorion (C), perivitelline space (PvS).
Figure D is a light micrograph that illustrates a metaphase mitotic figure found in the blastomeres prior
to the 4-cell stage (200x, PS). Figure E is a higher magnification view of the cleavage furrow separating
the two blastomeres (480x, SEM).
11
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Clf
Stage 5: Four-celled blastodisc. Plate 5
A second meridional cleavage cuts across the plane of the first at right angles and creates four equal-
sized globular blastomeres (B) with their lower surface formed by the yolk (Y) below. This cleavage also
takes about 7 minutes at 25°C. Some cytoplasm (Cy) in the yolk is still visible. The embryo rotates
freely within the chorion. Figure A shows the four globular blastomeres and the cleavage furrow (C1F)
separating them (80x, SEM). The four blastomeres are visible on the yolk mass in Figure B (80x, LS).
Figure C is from a serial section and illustrates the cleavage planes that separate the four blastomeres
(90x, PS). The dashed line in Figure D indicates the plane of section for Figure C (80x, LD); chorion
(C), perivitelline space (PvS).
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CIF--
Stage 6: Eight-celled blastodisc.
Plate 6
Four synchronous meridional cleavages occur parallel to the first cleavage plane, resulting in 8 equal-
sized blastomeres (B) arranged in 2 rows of 4 cells each. Minor irregularities in this arrangement are not
uncommon. Small yolk granules are visible in the blastodisc cytoplasm adjoining the yolk mass (Y) and
extending upward along the cleavage planes. Immediately underlying the blastodisc, yolk particles
appear smaller. Cleavage planes cut completely through the blastodisc to the surface of the yolk. Figure
A is a dorsal view showing the two parallel rows of four blastomeres (80x, SEM). Figure B offers a side
view of four of the eight blastomeres on the yolk mass (80x, LS). Figure C is a serial section showing
four blastomeres on the yolk mass. Two of the blastomeres contain distinctive mitotic figures (Mt) (80x,
PS). Figure D shows the large perivitelline space (PvS) which is present under the chorion (C) and a
limited amount of cytoplasm (Cy) in the yolk (80x, LD).
13
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Stage 7: Sixteen-celled blastodisc.
Plate?
Cleavage planes are parallel to the second cleavage and at right angles to the first and third. Cleavage
is synchronous and generally results in 4 parallel rows of 4 blastomeres (B) of approximately equal size.
Cleavage furrows at this stage do not cut completely through the cytoplasm, but leave a continuous layer
of cytoplasm adjoining the yolk (Y). This layer of syncytial cytoplasm is the presumptive periblast layer.
Figure A shows the blastomeres organized in 4 rows, each consisting of 4 blastomeres (80x, SEM).
Figure B is a micrograph of a dechorionated live embryo (80x, LS). Figure C shows the chorion (C),
perivitelline space (PvS), blastomeres, as well as the residual cytoplasm (Cy) in the yolk (80x, LD)
14
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Stage 8: Thirty-two-celled blastodisc.
Plate 8
Cleavages at this stage start to become asynchronous and difficult to follow. The cleavage planes are
no longer strictly perpendicular to yolk mass. Blastomeres (B) are 2 cells deep in the center of the
blastodisc. No intercellular spaces are present between blastomeres. The periblast layer of syncytial
cytoplasm has migrated from under the blastodisc to form a limited periblast corona. The chorion (C) is
now water-hardened and averages 1.4 mm in diameter. Figure A shows approximately 32 blastomeres
that form a mound on the yolk mass (Y) (80x, SEM). Figure B is a micrograph of a live dechorionated
embryo (80x, LS). Figure C shows the last remnant of cytoplasm (Cy) visible on the yolk mass (80x,
LD); perivitelline space (PvS).
15
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Stage 9: Late cleavage.
Plate 9
The individual blastomeres form an elevated cap upon the yolk mass. Spaces between the blas-
tomeres are now present. These spaces later become continuous and form the small ventral segmentation
cavity or blastocoele. The closely packed outermost layer or envelope of blastomeres form the early
epidermal stratum (ES) which surrounds the loosely packed inner cells. The periblast corona (PC) has
enlarged. Large yolk particles are visible in the cytoplasm of the deep-lying blastomeres. Figure A shows
the early epidermal stratum, the surface layer of blastomeres (80x, SEM). In Figure B note the lack of
cytoplasm visible in the yolk (Y) (80x, LS). Figure C is a serial section that shows the individual blas-
tomeres and small yolk particles (YP) within their cytoplasm (80x, PS). Figure D illustrates the limited
periblast corona extending from the blastomeres and the surface cells of the epidermal stratum. Plane of
section for Figure 9C is indicated by dashed line (80x, LD); chorion (C), perivitelline space (PvS).
16
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10
Stage 10:Highblastula.
Plate 10
The epidermal stratum (ES) is distinct and surrounds loosely packed globular interior cells. The
blastomere nuclei are large and distinct. The blastoderm rests on the periblast. The periblast corona (PC)
has expanded and covers approximately one-fifth of the yolk mass (Y). Periblast nuclei appear to arise at
the edge of the blastoderm from divisions of the marginal cells, although mitosis in isolated periblast
nuclei is also visible. Large yolk particles appear to be excluded from the blastoderm by the periblast
layer. Spaces between blastomeres have increased to form the low broad segmentation cavity (Sg),
which is bordered dorsally by globular cells of blastoderm and ventrally by the periblast layer. One
portion of the periblast corona has become thickened (TP) and lies immediately adjacent to a thickened
region of the blastoderm, where the embryonic shield will form. Microvilli remain present on the plasma
membrane of the blastomeres.
Figure A is a serial section that shows the epidermal stratum, consisting of blastomeres (B) which are
more squamous in appearance than the deeper-lying blastomeres. The segmentation cavity and the
region of thickened periblast are also easily seen (80x, PS). The early periblast corona is seen in Figure
B (80x, LS). The periblast corona is more clearly shown in Figure C. The periblast corona has enlarged,
while the blastomeres have decreased in size from the previous stage (80x, LD). Figure D shows the
epidermal stratum and the underlying yolk mass (120x, SEM). Figure E is a higher magnification micro-
graph of the blastomeres of the epidermal stratum which are covered with numerous microvilli (Mv). A
cleavage furrow (GIF) between two blastomeres is also visible in this figure (7400x, SEM); chorion (C),
perivitelline space (PvS).
17
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11
0
Stage 11: Flat blastula. Plate 11
The blastoderm forms a flattened lens-shaped structure called the flat blastula, which caps the yolk
mass (Y). There is a further reduction in size, and an increase in number of individual blastomeres.
Extensive mitotic activity is visible within the blastomeres and in the periblast nuclei. Cells of the
epidermal stratum (ES) are becoming squamous. It is often difficult to distinguish the flat blastula from
the early gastrula states in living embryos. The broad segmentation cavity (Sg) or blastocoele is still
present.
Figure A shows the small size of the individual blastomeres. Under poor lighting conditions it may be
possible to mistake embryos of the high or flat blastula stages for one-celled embryos (80, LS). Figure B
is a line drawing that illustrates the further increase in size of the periblast corona (PC) and decrease in
size of the blastomeres (66x, LD). Figure C is a serial section that shows mitotic figures in the region of
thickened periblast (TP). Also note the epidermal stratum and the distinct nucleus (Nu) of an inner
blastomere (200x, PS). Figure D is a serial section that shows the location of the segmentation cavity
and the thin layer of underlying periblast (P) (80x, PS); chorion (C), perivitelline space (PvS).
18
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12
Stage 12: Early gastrula. Plate 12
At the onset of gastrulation, the edge of the blastoderm (Bl) becomes elevated to form the early germ
ring (GR), which results in a slight depression in the yolk mass (Y). The embryonic shield anlage is
faintly visible as an enlargement of one portion of the germ ring that is preceded by a region of thick-
ened periblast. Much mitotic activity is visible in this thickened periblast layer. The number of cells
within the central blastoderm has decreased and the segmentation cavity has enlarged.
Figure A shows the slightly thickened profile of the germ ring which forms the advancing edge of the
blastoderm (80x, PS). Note much yolk has been lost during sectioning. Figure B shows the flattened
mass of cells of the blastoderm that have started to migrate over the yolk mass (80x, LS). Figure C
shows the periblast corona (PC) which continues to enlarge and migrate down over the yolk mass (80x,
LD); epidermal stratum (ES), thickened periblast (TP), chorion (C), perivitelline space (PvS).
19
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13
.Gt
Stage 13: One-quarter epiboly. Plate 13
The germ ring (GR) has migrated one-quarter of the way over the yolk mass (Y) and has thickened to
become elevated above the blastodisc. Much mitotic activity is evident in the blastomeres (B). An exten-
sive segmentation cavity (Sg) is still present. The periblast corona (PC) is clearly visible in living em-
bryos. Figure A shows the segmentation cavity which is bordered ventrally by the periblast layer and
dorsally by the blastomeres. Considerable yolk has been lost during the preparation of this section (80x,
PS). Figure B shows the germ ring visible as the edge of the migrating blastoderm (80x, LS). Figure C
shows the thickened germ ring, the chorion (C), periblast corona, yolk mass (Y), and perivitelline space
(PvS) (80x, LD).
20
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14
.„
EmS -
IP -
'
, :
•** Y
CIF-
EmS
Stage 14: One-half epiboly.
Plate 14
Epiboly continues with the germ ring (GR) covering one-half of the yolk mass (Y). The inner layer of
cells in the germ ring is termed the hypoblast, which is formed by the outward movement of inner cells
of the blastoderm (Bl) (Ballard, 1966). The central area of the blastoderm continues to thin. Mitotic
figures are no longer visible in the periblast layer, but are evident in the blastomeres. The large oval
periblast nuclei are visible in the thickened periblast (TP) layer preceding the embryonic shield (EmS).
The embryonic shield appears as a thickened region of the germ ring, slightly closer to the animal pole
than is the surrounding germ ring.
Figures A (80x, SEM) and B (80x, LS) show the germ ring and the localized elevated region of
blastomeres that make up the embryonic shield . Figure C illustrates the germ ring, embryonic shield,
periblast corona (PC), chorion (C), and perivitelline space (PvS) (80x, LD). Figure D shows the embry-
onic shield and associated region of thickened periblast (80x, PS). Figure E shows the wrinkled appear-
ance of the surface blastomeres. Note the lack of microvilli (3000x, SEM). Figure F is from a serial
section that shows the region of thickened periblast with its periblast nuclei (PN) preceding the embry-
onic shield (200x, PS).
21
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15
Stage 15: Three-quarters epiboly. Plate 15
The germ ring now covers three-quarters of the yolk mass. An unlined vesicle (V) in the periblast (P)
under the advancing edge of the embryonic shield is visible in serial sections and creates a depression in
the yolk mass. A streak of neuroectoderm cells is behind the embryonic shield. Prior to stage 16, this
streak of cells thickens and forms the neural keel anlage. The neural keel, as in other teleosts, forms as a
solid mass of neuroectoderm, with the neurocoele forming later in development. The yolk plug (YP1)
protrudes slightly from the edge of the advancing germ ring. Mesodermal cells of the somite anlagen
(SA) have aligned themselves lateral to the neural keel. Many periblast nuclei (PN) are visible in the
periblast layer under the presumptive caudal knob (CK).
Figure A is a sagittal section that shows where the anterior (A) and caudal (Ca) regions of the embryo
will form (80x, PS). Figure B shows the thickened ridge of cells that make up the embryonic shield (ES).
At this stage the yolk plug has not been covered by the advancing germ ring (80x, LS). Figure C illus-
trates the future caudal end of the embryo. The plane of section of Figure E is indicated by a dashed line.
The periblast corona (PC) almost completely covers the yolk plug (80x, LD). Figure D is a serial section
that shows an unlined vesicle (V) and the underlying periblast nuclei in the thickened periblast (TP)
(400x. PS). Note the appearance of the yolk mass (Y). Figure E is a serial section that shows the area of
the presumptive caudal knob, as well as the lateral somite anlagen. The unlined vesicle and surrounding
thickened periblast and periblast nuclei are also visible (lOOx, PS).
22
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16
Stage 16: Closure of the germ ring. Plate 16
This stage marks the end of epiboly with the closure of the germ ring (GR) over the yolk plug. This
process is not synonymous with the closure of the blastopore in amphibians. An elevated cluster of cells
is present at the caudal end (Ca) of the embryonic axis where the germ ring closed. A depression in the
yolk mass (Y) is visible immediately below this caudal knob (CK). The periblast nuclei continue to
proliferate in the periblast layer (P) under the caudal knob. The optic anlagen are visible as two lateral
enlargements of head region (HR) which appears as a cluster of cells at the anterior end of the embryo.
Anterior and caudal cell clusters are connected by a low broad ridge of cells, the presumptive neural
keel. A group of undifferentiated mesodermal cells that form the presumptive notochord (N) are present
ventral to this neural keel anlage. There is a migration of lateral mesodermal cells toward the embryonic
axis. These cells constitute the somite anlagen (SA).
A thin layer of cells made up externally of the epidermal ectoderm layer which has differentiated
from the epidermal stratum and an undifferentiated layer of mesodermal cells internally have lifted free
of the yolk mass anterior to the head region to form the precursor to the pericardial coelom. Figure A
shows the elevated caudal knob and the head region of the embryo. Note the large fluid-filled presump-
tive pericardial coelom (PCo) (80x, LS). In Figure B the plane of section for Figure D is indicated by a
dashed line. Note the site of closure of the germ ring (66x, LD). Figure C is a serial section that shows
the elevated undifferentiated mass of neuroectoderm in the head region (80x, PS). Figure D is a trans-
verse serial section that shows the undifferentiated mesodermal cells of the lateral somite anlagen that
flank the notochord (lOOx, PS); chorion (C), perivitelline space (PvS).
23
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o»
D s
Stagel7:Neurula. Plate 17
The prosencephalon (Pr), mesencephalon (Me), and rhombencephalon (R) regions of the brain can be
distinguished at this stage. The neural keel consists of a solid wide band of cells that extend caudally
from the rhombencephalon. The notochord is visible as a solid rod of mesodermal cells ventral to the
neural keel and extends from the rhombencephalon to the undifferentiated caudal knob (CK). The
notochord is flanked laterally by mesodermal cells which have differentiated into 4-5 pairs of somites.
Kupffer's vesicle (KV) is present as an oval-shaped vesicle ventral to the caudal knob, lined with colum-
nar epithelial cells. Optic anlagen (OA) are visible as lateral swellings of the presumptive diencephalon.
The infundibulum anlage starts to form from a ventral evagination of the presumptive diencephalon.
Thin sheets of slightly darker-staining neural crest cells are visible both dorsally and ventrally on the
lateral aspects of the optic anlagen.
Figure A is a sagittal section that illustrates the optic anlage and the somites (80x, PS) Again note the
loss of yolk material from the yolk mass (Y) during sectioning. Figure B shows the three regions of the
brain: the prosencephalon, mesencephalon, and rhombencephalon. Also note the anterior-most presump-
tive pericardia! coelom (PCo) (80x, LS). Figure C is a line drawing that shows the three brain regions,
prosencephalon, mesencephalon, rhombencephalon, the caudal knob, Kupffer's vesicle, chorion (C),
perivitelline space (PvS) and four somites. The plane of section for Figure D is indicated by a dashed
line (80x. LD). Figure D is a transverse serial section that shows the optic anlage as lateral evaginations
of the prosencephalon. The presumptive infundibulum is present as a ventral evagination of the early
diencephalon. Also note the paired block-like caudal somite anlage (80x, PS). In Figure E Kupffer's
vesicle bounded by columnar epithelial cells is visible above the thick region of caudal periblast (P).
Note the large periblast nuclei (PN) in the caudal periblast (400x, PS).
24
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18
NCr
ov
M.
.OV
—PCo
OV
: '•
'—CK
Stage 18: Optic vesicles.
Plate 18
The site of invagination of the optic vesicles in live embryos appears as a horizontal slit on their
lateral aspects. The prosencephalon and optic vesicles are arrowhead-shaped when viewed from above.
A depression is forming on the dorsal aspect of the brain between the mesencephalon (Me) and the
rhombencephalon. Rudiments of the first and second ventricles of the forebrain are visible as small
lateral slits in the anterior end of the prosencephalon. The third ventricular rudiment is a narrow dorsal-
ventral slit which is continuous with narrow cavities in the optic vesicles (OV). Cells of the solid neural
keel (NK) have become aligned parallel to the yolk mass and have segregated into two lateral masses,
although no neurocoele is present. The neural keel is oval in cross-section anteriorly and becomes
triangular caudally. A large infundibulum extends ventrally from the diencephalon (D). Cells of the
notochord (N) are elongating and becoming vacuolated.
Caudally, the notochordal cells become increasingly disorganized until they grade into undifferenti-
ated mesoderm of the caudal knob (CK). Several unlined vesicles (V) are present in the thick periblast
layer (P) ventral to Kupffer's vesicle (KV). The number of periblast nuclei in the caudal periblast contin-
ues to increase. The pericardial coelom (PCo) is present as a fluid-filled sac anterior to and ventral to the
first one-third optic vesicles. Nine to ten pairs of somites (S) are present. Somites are 6-7 cells thick
adjacent to the notochord, but taper off into lateral plate mesoderm laterally and into flattened segmental
mesoderm (SM) caudally. A small coelomic cavity (Co) is present in the segmental mesoderm. There is
no indication of circulatory or digestive system rudiments. Neural crest cells (NCr) are visible as diffuse
masses of cells dorsal-lateral and ventral-lateral to the diencephalon and mesencephalon, and as diffuse
masses of cells lateral to the nerve cord in mid and caudal regions. Figure A is a transverse section that
shows the optic vesicles situated lateral to the diencephalon. Note the lack of neurocoele in the dien-
cephalon. Neural crest cells are visible dorsal and lateral to the diencephalon. Plane of section is repre-
sented by the dashed line (a) in Figure C (200x, PC). Figure B illustrates the optic vesicles, pericardial
-------
coelom, caudal knob, somites and translucent yolk mass (Y) in a live embryo (80x. LS). Figure C is a
line drawing that illustrates the optic vesicles, caudal knob, notochord, and somites (80x, LD). In Figure
D the triangular neural keel is visible above the notochord. A small coelomic cavity is visible in the
segmental mesoderm. Plane of section is indicated by dashed line (d) in Figure C (200x, PS). Figure E
shows Kupffer's vesicle and an unlined vesicle extending into the caudal periblast and yolk mass (400x,
PS).
26
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19
OiP
EE -
Stage 19: Neuromeres.
Plate 19
Neuromeres (Nm) are visible as a series of small enlargements or evaginations in the dorsal myelen-
cephalon. A neurocoele is present only in the form of the reduced brain ventricles. The cavity within the
optic cups (OC) has increased in size. The ventrally located pericardial coelom (PCo) has enlarged both
anteriorly and posteriorly. The otic placodes (OtP) are visible as a pair of lateral thickenings in the
epidermal ectoderm (EE) in the rhombencephalic region. The 14 pairs of somites (S) are bounded by a
layer of columnar cells and have condensed to form more block-shaped segments. Notochordal (N) cells
are becoming increasingly vacuolated, and the notochord has increased in length anteriorly. A thick
periblast layer with its nuclei and Kupffer's vesicle (KV) are present below the somewhat flattened
caudal knob. The embryo is bounded by a squamous epithelial layer of ectoderm. Neural crest cells
(NCr) continue to migrate around the brain and nerve cord (NC).
In Figure A the otic placodes are visible in this sagittal section as thickenings in the epidermal ecto-
derm. Note that Kupffer's vesicle and the optic cup are still prominent structures (80x, PS). In Figure B a
large optic cup is visible in the prosencephalon. The otic placode is visible as a slightly clear region in
the rhombencephalon (80x, LS). In Figure C neuromeres are visible as a series of small evaginations in
the dorsal rhombencephalon. Fourteen somites are shown as are the distinctive optic cup and prosen-
cephalon. Planes of section for Figures D and E are indicated by dashed lines (d) and (e), respectively
(80x, LD). Figure D is a transverse serial section that shows the solid nerve cord posterior to the
rhombencephalon. The notochord and Kupffer's vesicle are also visible in the caudal region (80x, PS).
In Figure E the posterior edge of the optic cup is visible, as is its connection with the diencephalon (D).
Neural crest cells are visible as dorsal and ventral masses of cells lateral to the diencephalon (150x, PS).
Note that this stage and all subsequent stages show figures of dechorionated embryos.
27
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20
OlV
re.
Stage 20: Otic vesicles.
Plate 20
Otic vesicles (OtV) have formed, and each possesses a small lumen surrounded by columnar cells
with peripherally located nuclei. The three primary brain regions and neuromeres are easily distin-
guished in living embryos. The mesencephalon (Me) is markedly elevated above the rest of the brain. 16
pairs of somites (S) are becoming chevron-shaped. The periblast nuclei are moving anteriorly out of the
caudal periblast layer. The pericardial coelom (PCo) continues to migrate posteriorly.
In Figure A the otic vesicle is visible as a clear region lateral to the rhombencephalon. The mesen-
cephalon is elevated above the rest of the brain. Sixteen somites, the notochord (N), the diencephalon
(D), telencephalon (Te), and pericardial coelom are also visible (80x, LS). Figure B shows the location
of Kupffer's vesicle (KV) as well as the other structures identified in Figure A (80x, LD). In Figure C the
small lumen and columnar cells that make up the otic vesicle are visible in sagittal section (200x, PS). In
Figure D Kupffer's vesicle and an unlined ventral vesicle (V) are visible in the caudal region. The
embryo is bounded by the epidermal ectoderm layer (EE) (400x, PS).
28
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21
Stage 21 :Tailbud.
Plate 21
The tailbud (TB) has formed and is starting to lift free of the yolk mass (Y). The 18-20 pairs of
somites (S) are becoming increasingly chevron-shaped. A fissure (MF) separating the mesencephalon
(Me) and the metencephalon has formed perpendicular to the embryonic axis. The aqueduct of Sylvius
connects the slightly enlarged third ventricle (3v) with the fourth ventricle (4v). The spinal cord is oval
in transverse section and contains a narrow neurocoele. The olfactory placodes (O1P) are visible as a
pair of thickenings in the ectoderm between the optic cups (OC). The lens placodes (LP) are present as
rounded masses of undifferentiated ectodermal cells, extending into the optic cup but still attached to the
epidermal ectoderm (EE). The periblast under the tailbud has become reduced in size. Periblast nuclei
continue to migrate anteriorly in the periblast or yolk-sac syncytium. There is no indication of the gut
anlage at this stage. A broad ventral mass of mesoderm extending caudally from the optic cups will form
the heart anlage in later stages. The pericardial coelom (PCo) continues to migrate posteriorly.
Figure A shows the tailbud, enlarged otic vesicle (OtV), optic cup, and the mesen-metencephalon
fissure (MF). Note the increase in somite number since the previous stage (80x, LS). In Figure B the lens
placode which is not visible in Figure A is illustrated in this line drawing. The plane of section of Figure
D is indicated by the dashed line (80x, LD). Figure C is a sagittal section that shows how far posterior
the pericardial coelom is located. Also note the elongated cells of the notochord (80x, PS). Figure D is a
transverse section that shows the lens placode still attached to the epidermal ectoderm. Scattered neural
crest cells are visible lateral to the diencephalon. Note the enlarged third ventricle and the scattered
neural crest cells (NCr) on the lateral aspects of the diencephalon (D) (120x, PS).
29
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22
O'V
Stage 22: First movements. Plate 22
Weak twitching movements are first observed in living embryos. The tailbud (TB) has elongated and
is free of the yolk mass (Y). Brain ventricles and the neurocoele (Nc) of the nerve cord (NC) have
enlarged. The olfactory placodes (O1P) have sunk into to epidermal ectoderm (EE) to form the olfactory
pits which are visible in living embryos. The otic vesicles (OtV) have enlarged. The lens has formed and
is clearly visible in the optic cups, which remain connected to the diencephalon (D) by a hollow cord of
cells. Muscle fiber cell precursors in the 20 somite (S) pairs are elongating and becoming multinucle-
ated. The pericardia! coelom (PCo) has enlarged considerably. The heart anlage (HA) is visible as an
anterior-ventral evagination from a broad sheet of mesoderm located under the mesen-metencephalic
fissure (mesen-metencephalic fold). The embryonic coelom is visible as a small cavity in the mesoderm
lateral to the somites. Neuromast anlagen are visible as columnar thickenings of cells in the epidermal
ectoderm lateral to the nerve cord in the midregions of the embryo.
Figure A clearly illustrates the enlarged tailbud and pericardial coelom (80x, LS). Note the appear-
ance of the somites and notochord (N). In Figure B the olfactory placode is indicated by a line in the
anterior telencephalon. The lens (L) and otic vesicle (OtV) have enlarged and are easily distinguished in
living embryos. The plane of section for Figure C is indicated by a dashed line (80x, LD). In Figure C
the enlarged lens is visible in the optic cups. Note the large neurocoele in the nerve cord (80x, PS).
Figure D is a sagittal section that shows the enlarged lumen of the otic vesicle. The heart anlage is
visible as two sheets of mesoderm, the early endocardium and epimyocardium layers. The olfactory
placode is visible as a thickening in the epidermal ectoderm in the anterior telencephalon; yolk (Y)
(200x, PS).
30
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23
oiv
oiv
Stage 23: Heartbeat.
Plate 23
The S-shaped tubular heart lies in a depression in the yolk (Y) ventral to the left eye. A colorless fluid
is visible moving in the heart as it beats sporadically, although no peripheral circulation is present. Blood
islands are forming as clumps of cells on the yolk mass. The embryo exhibits extensive tail thrashing
movements. The brain ventricles have enlarged, and the roof of the rhombencephalon has started to
become thinner. The mesen-metencephalon fissure (MF) is more pronounced than previously. The large
infundibulum (I) has expanded laterally. The lens (L) and the ventral choroid fissure (CF) are distinct in
living embryos. Periblast nuclei continue to migrate laterally and anteriorly from the periblast region
under Kupffer's vesicle (KV). In the caudal region, the dorsal and ventral medial fin folds are visible as
thickened evaginations in the epidermal ectoderm. Muscle fibers remain immature, but continue to
elongate. The olfactory pits are lined with columnar epithelial cells and are located medial to the optic
cups (OC). Paired pronephric ducts are present in the caudal region as a pair of thin-walled laterally
located tubes, each having a small lumen. They merge anteriorly to form a single thin-walled structure,
the urinary bladder anlage.
Figure A shows the large pericardial coelom (PCo) and otic vesicle (OtV). The yolk mass is consider-
ably reduced in size compared to the rest of the embryo (80x, LS). In Figure B the ventral choroid
fissure and enlarged somites (S) are visible. The mesen-metencephalic fissure and otic vesicle are easily
distinguished in living embryos. The plane of section of Figure C is indicated by the dashed line (80x,
LD). In Figure C the large lens is visible in the optic cup. The infundibulum has expanded laterally. The
ventral nerve cord (NC) and dorsal notochord (N) are visible in the caudal region (80x, PS). Figure D is
a sagittal section that shows the somites. Their distinctive appearance is due to the elongated muscle
fibers. The optic cup, lens, and otic vesicle are clearly visible. Note the large Kupffer's vesicle and the
vacuolated cells of the notochord (80x, PS).
31
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24
'
Stage 24: Circulation. Plate 24
Blood islands resting on the yolk mass (Y) consist of light yellow aggregations of cells. These cells
are being washed into the large common cardinal veins (cc) by the blood flow. Colorless nucleated blood
cells leave the heart (H) and enter the ventral aorta which divides anteriorly into the internal carotid and
hyaloid arteries, and posteriorly to form the dorsal aorta. The large caudal artery (CA) lies immediately
under the notochord (N) and extends to the sixth precloacal somite (S). The caudal vein (CV) is immedi-
ately ventral to the caudal artery. Otoliths are visible as two small concretions in each otic capsule (OtC).
The lens (L) has enlarged and is surrounded by a single layer of columnar epithelium. The dorsal and
ventral median fin folds continue to form anteriorly. The gut can be seen as a thin-walled ventral tube of
endoderm extending from the mesencephalon to the proctodeum. The proctodeum is visible as an invagi-
nation in the epidermal ectoderm ventral to the terminus of the hindgut. The liver diverticulum (LDi) is
present as a mass of tissue budding off from the ventral foregut below the posterior myelencephalon.
The paired pronephric ducts (PT) are ventral and lateral to the caudal vein, and are bounded by a
single layer of columnar cells. Neuromasts (Nmt) of the lateral line system are visible as condensations
of tissue under the epidermal ectoderm lateral to the somites (S). The tail has elongated and the embryo
now possesses 35 to 37 pairs of somites. A lighter-staining region (white matter) consisting of axons is
visible on the lateral aspects of the diencephalon (D) medial to the optic cups (OC). The third and fourth
ventricles have enlarged. Cells of the notochord (N) are highly vacuolated. The notochord is encased in a
thin hyaline sheath (HS).
In Figure A the distinctive third (3v) and fourth (4v) brain ventricles are easily seen in living embryos
(80x). In Figure B the otoliths are visible as a pair of small concretions in the otic vesicle. The liver
diverticulum appears as a depression in the yolk sac. The olfactory groove (OG) is visible in the anterior
telencephalon. Planes of section for Figures C and D are represented by dashed lines (c) and (d), respec-
tively (80x. LD). In Figure C the heart is visible as a thin tube of mesoderm under the optic cup. The
32
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lens is surrounded by columnar epithelium and infundibulum (I) (80x, PS). Figure D is a transverse
serial section that shows the spinal cord (SC), the caudal artery and vein and the notochord which is
encased in a hyaline sheath. The lens-shaped neuromasts are visible lateral to the caudal artery (200x,
PS). Figure E is a sagittal serial section that shows the optic cup, third and fourth ventricles, and heart
(200x, PS).
33
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25
00
Stage 25: Retinal pigmentation.
Plate 25
The outermost layer of flattened epithelial cells surrounding the optic cup has become pigmented,
giving the optic cups a darkened color. The head is beginning to lift free of the yolk mass. The marked
cephalic flexure occurs at the mesen-metencephalic transition. The optic nerve (ON) leaves the optic cup
through the ventral choroid fissure (CF). The white matter (WM) continues to increase in volume on the
ventral surface of the brain and spinal cord. The tectal lobes (TL) are visible as evaginations on the
dorsal and lateral surfaces of the mesencephalon. On the ventral surface of the anterior yolk mass, a
large sac of blood is filled by the cardinal veins and drained by the sinus venosus. Blood islands are still
present on the yolk sac. The heart (H) has a definite S-shape and lies primarily on the yolk sac under the
right optic cup. Blood flow extends to the third postcaudal somite (S). Small thickenings in the epider-
mal ectoderm (EE) are located throughout the head region and surrounding the cloacal opening. The
median fin fold (MFF) continues to form anteriorly.
The lens-shaped neuromasts of the lateral line system are visible at intervals below the epidermal
ectoderm lateral to the somites in the caudal region. Masses of mesenchyme and neural crest cells are
condensing lateral and ventral to the rhombencephalon near the otic capsule. The anterior pituitary
anlage is visible as an undifferentiated mass of ectodermal cells budding off the dorsal pharynx. Pharyn-
geal pouches 5 and 6 are visible as lateral pockets in the pharyngeal region anterior to the otic capsule.
Axons extend from a placode posterior to the optic cups toward the mesencephalon, and will later
become part of cranial ganglion V. The thin roof of the rhombencephalon which is the site of formation
of the choroid plexus is clearly visible in living embryos. Liver (Li) tissue continues to proliferate.
Embryos are quite active, exhibiting frequent thrashing movements.
In Figure A the median fin fold is easily visible in living embryos. The somites, heart, liver, and
otoliths (O) continue to differentiate. The choroid fissure is clearly visible in the optic cup. The plane of
section for Figure C is indicated by the dashed line (80x, LD). In Figure B the olfactory groove (OG) is
34
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visible in the anterior telencephalon. The otoliths appear as dark concretions in the otic capsule (80x,
LS). Figure C is a transverse section that shows the lateral tectal lobes and the thin layer of white matter
(200xPS). Figure D is a sagittal serial that shows the posterior choroid plexus which forms the roof of
the rhombencephalon. The optic nerve is visible as a lightly stained region ventral to the optic cup. Liver
tissue forms a depression in the yolk mass. Note the distinctive olfactory groove (OG) (lOOx, PS).
35
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Stage 26: Intersegmentai arteries. Plate 26
The two pectoral fin (PF) buds which first became visible at about 45 hr have enlarged. Blood flow is
seen for the first time in the intersegmental arteries. Blood cells are becoming more pigmented, and
blood flow now extends to the fourth postcaudal somite (S). The cardinal veins are well-defined and lie
in depressions in the shrinking yolk sac. The hindgut (G) consists of a small lumen surrounded by a
single layer of columnar epithelium and is located immediately ventral to the caudal vein. The lumen of
the hindgut enlarges just anterior to the cloaca. The sinus venosus, atrium, ventricle, and conus arteriosus
of the heart can be distinguished in living embryos. Powerful ventricular contractions cause the head of
the embryo to bob up and down. Circulation has increased in the head plexus. The eyes have a silvery
color. The embryo continues to elongate as the head lifts free of the yolk sac.
The enlarged olfactory grooves (OG) are lined by a layer of columnar cells. The epiphysis anlage
appears as a small evagination from the roof of the diencephalon. White matter (WM) continues to
increase in volume on the ventral and lateral surfaces of the brain and spinal cord (SC). Kupffer's vesicle
persists anterior to the cloaca. The highly vacuolated cells of the notochord (N) are transparent and
stacked in layers. The notochord tapers and terminates ventral to the anterior end of the otic capsule. The
otic capsule is no longer oval; instead, it is narrower at its base where the enlarged otoliths (O) are
located.
There is no indication of a stomodeum at this stage, but the pharynx has enlarged laterally. The liver
continues to enlarge. Muscle fibers continue to elongate and are developing the banding pattern typical
of striated muscle. Neuromasts have continued to proliferate and to migrate anteriorly. The eyes are
connected to the diencephalon by the optic nerve. A small fold of cells connects the spinal cord with the
dorsal fin fold. The pituitary anlage is visible as a compact mass of cells ventral to and closely appressed
to the large infundibulum. The squamous epidermal-ectodermal (EE) cells are pentagon-shaped.
Figure A is a SEM micrograph that shows a pectoral fin with its surface covered by epidermal ecto-
36
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derm cells (540x, SEM). Figure B is a line drawing that illustrates those structures easily seen in living
embryos and in serial sections: median fin fold (MFF), proctodeum (Pt), heart, common cardinals (CC),
otoliths, olfactory groove. The plane of section of Figures D and E are shown as dashed lines (d) and (e),
respectively (80x, LD). In Figure C the median fin fold, otoliths, common cardinals, olfactory groove,
pectoral fins, fourth ventricle (4v) and optic cup are visible (80x, LS). Figure D is a frontal serial section
that shows the lens in the optic cup (OC) as well as the large olfactory grooves in the epidermal ecto-
derm (200x, PS). Figure E shows white matter on the ventral and lateral surfaces of the spinal cord. Note
the pectoral fins, notochord, caudal artery (CA), liver tissue (Li), and gut with a small lumen (200x, PS).
37
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27
Stage 21: Horizontal duct.
Plate 27
The walls of the otic or auditory capsule (OtC) have thickened ventrally and thinned dorsally where
the horizontal duct is forming. The paired otoliths (O) are oval-shaped and have become perpendicular in
orientation to each other. The olfactory grooves have elongated and are closely associated with the
anlage of the olfactory lobes, although no connection is visible yet.
A strong blood flow is present in the intersegmental arteries. A short vessel extends caudally beyond
the turnaround point of the caudal artery. Radial striations centered around the tip of the notochord (N)
have started to form in the caudal fin. The medial fin fold (MFF) continues to enlarge and extend anteri-
orly. The surface thickenings in the epidermal ectoderm noted in previous stages are darkening. The
notochord extends anteriorly to the mesen-metencephalic fissure and lies just dorsal to the pharynx. A
small unlined vesicle and scattered large periblast nuclei are present at the posterior end of the yolk sac
just anterior to the cloaca (Cl).
The presumptive cone cells have elongated inside the pigmented layer of the eye. The precursors of
cranial ganglia IX and X are visible in serial sections as dorsal-lateral placodes sending bundles of axons
toward the myelencephalon. White matter continues to increase in volume. The two lateral cavities of the
tectal lobes have enlarged in the mesencephalon. Neuromasts of the lateral line system are visible in
lateral pairs throughout the head region. The mandibular, hyoid, and pharyngeal arches 3 and 4 are
visible. Masses of tissue which later form the parachordal and trabecular cartilages are condensing
dorsal to the pharynx. The early elements of the lower jaw are visible in sections as lateral and ventral
condensations of tissue of the pharynx.
Figure A is a line drawing that illustrates the perpendicular orientation of the otoliths in the otic
capsule. The notochord, pectoral fins (PF), olfactory groove (OG), somites (S), and heart (H) continue to
differentiate. Plane of section of Figure D is indicated by the dashed line (80x, LD). In Figure B the
large heart is visible on the anterior yolk sac. The notochord, fourth ventricle (4v) and pectoral fins are
38
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easily seen in living embryos (80x, LS). Figure C is a sagittal serial section that shows white matter on
the ventral hind brain. The thick optic nerve (ON) and extensive liver (Li) are also visible. The epiphysis
is visible as an evagination in the roof of the diencephalon (lOOx, PS). Figure D is a transverse serial
section that shows the small medial fin fold as a fold in the epidermal ectoderm. The large somites are
lateral to the spinal cord and notochord. The caudal artery (CA) and caudal vein (CV) are located dorsal
to the small gut (G) (200x, PS).
39
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28
Stage 28: Blood in pectoral fins.
Plate 28
Blood flows through a single loop in the pectoral fins (PF). Dark stellate melanocytes are present on
the ventral surface of the yolk sac (Y) anterior to the cloaca (Cl). An enlarged vessel dorsal to the myo-
tomes receives blood from the segmental arteries. The six aortic arches branch off of the ventral aorta.
Ventral and lateral evaginations into the lumen of the otic capsule (OtC) have appeared. The liver (Li)
has noticeably enlarged. The thick-walled heart (H) lies completely on the yolk sac (Y). The angle of the
cephalic flexure has decreased. The hyaline sheath surrounding the notochord (N) has thickened. The
banding pattern of the skeletal muscle is more apparent.
Cranial ganglia V, IX, and X have connected with the diencephalon and myelencephalon. A bridge of
tissue representing the semicircular canal anlage is visible in the otic capsules. Neuromasts continue to
proliferate throughout the head region. The olfactory grooves have sent out axons which link up with the
olfactory lobes of the telencephalon. The operculum extends caudally from the hyoid arch and covers
branchial arches 3, 4, and 5. White matter extends along the dorsal and lateral sides of the spinal cord for
most of its length.
The optic cup (OC) has darkened considerably, obscuring much of the lens. The eyes are enclosed in
a 2-3 cell-thick layer of undifferentiated mesoderm, the early sclerotic coat (SC) which is enclosed the
by the pigment epithelium (PE). The retina itself has differentiated into a layer of columnar cone precur-
sor cells, a bipolar layer, and the thick ganglion cell layer (GL). The distinctive choroid fissure pierces
all layers of the eye, and is the route of exit for the optic nerve and entry for the hyoid artery. The thin
layer of epidermal ectoderm and mesoderm covering the eye has not yet differentiated into cornea.
The hindgut makes an abrupt ventral turn above the cloacal opening, then increases in size just prior
to merging with the opening of the urinary bladder anlage. Paired posterior pronephric ducts (PD) join
into a single enlarged duct lined with columnar epithelium, the urinary bladder anlage, which also makes
an abrupt ventral turn prior to merging with the small cloaca. Paired pronephric ducts extend anteriorly
40
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to the pectoral fins. Cartilaginous tissue is differentiating in the upper and lower jaws. The pharynx is
large and triangular anteriorly, becoming compressed dorsally and ventrally prior to merging with the
foregut (Fg). A bend in the foregut is visible under the pectoral fins. Radial cartilage (RC) is forming in
the pectoral fins.
Figure A is a line drawing that shows the cloaca which functions as the common exit for the digestive
and urinary system. The otic capsule, liver, somites (S), and heart (H) continue to differentiate. A small
vessel loops through the pectoral fin with a visible blood flow. The planes of section for Figures C and D
are indicated by dashed lines (c) and (d), respectively (80x, LD). In Figure B the liver is visible extend-
ing into the yolk sac of living embryos. The optic cups appear as dark masses due to extensive pigmenta-
tion. Note the thickened walls of the otic capsule (80x, LS). Figure C is an oblique serial section that
shows the lens and layers of the eye, the sclerotic coat, the pigment epithelium, the outer nuclear layer
(ONL), the inner nuclear layer (INL), internal plexiform layer, and the ganglion cell layer (200x, PS).
Figure D is a transverse section that shows the radial cartilage in the pectoral fin. The thick-walled
foregut is medial to the liver tissue. Paired pronephric ducts are ventral and lateral to the notochord
(200x, PS).
41
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29
Stage 29: Bile formation. Plate 29
Further differentiation has occurred in the cartilage of the jaw apparatus. Limited movement of the
lower jaw and eyes in living embryos is first seen at this stage. Yellowish bile in the gall bladder has
formed and is visible in living embryos. A dip is present in the median fin fold (MFF) around the cloacal
(Cl) opening. The number of stellate melanocytes has increased on the ventral yolk sac (Y). The caudal
artery has replaced the posterior dead-end vessel of the previous stage. Striated muscle and basal carti-
lage is present in the base of the pectoral fins (PFO). The ampullae anlagen at the base of the semicircu-
lar canal are visible as thickenings in the walls of the otic capsule (OtC). The foregut (Fg) has differenti-
ated into a thick-walled stomach with an enlarged lumen. Blood flow can now be observed in the liver
(Li) sinusoids. The olfactory grooves (OG) are bordered internally by a squamous cell layer, and exter-
nally by a lighter-staining sensory layer.
Figure A is a line drawing that illustrates the lower jaws (LJ) and the continued differentiation of the
otic capsule. Also visible are the notochord (N), heart (H), common cardinal veins (CC), third (3v) and
fourth (4v) ventricle, choroid fissure, somites (S), and median fin fold. The plane of section of Figure D
is indicated by the dashed line (80x, LD). In Figure B the otic capsule and the highly pigmented optic
cup (OC) are seen (80x. LS). Also note the heart on the anterior aspect of the yolk mass. Figure C is a
sagittal serial section that shows the thick-walled foregut and liver tissue. Four aortic arches (AA) are
visible in the branchial arches (80x, PS). Figure D is an oblique serial section that shows the optic nerve
in the optic cup. The large olfactory groove, head ectoderm (HE), and pharynx (Ph) are also visible
(lOOx. PS).
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30
Stage 30: Swim bladder.
Plate 30
At this stage embryos hatch out of the chorion if disturbed by handling or bright lights. The swim
bladder (SB) which appeared at 90 hr (at 25°C) has enlarged and is pigmented dorsally. It maintains its
connection with the foregut through a thin hollow cord of cells. The stomach (St) is thick-walled and is
developing internal folds. The lumen of the gall bladder (GB) is lined with a single layer of columnar
cells, and is connected to the intestine by a short bile duct. The yellow bile from the bile duct (BD) is
visible, extending caudally into the intestine.
The eyes, jaws, pectoral fins (PF), and operculum (Op) exhibit frequent movements. The mandibular
adductor and levator muscles have formed in the head region in association with the protruding lower
jaw (LJ). The mouth is now open. The epidermal ectoderm (EE) covering the shrinking yolk sac (Y) has
a wrinkled appearance. The somites (S) extend to the posterior edge of the otic capsule (OtC). Increased
pigmentation is visible on the ventral yolk sac. Melanocytes are present at intervals along the lateral line
system associated with the neuromasts. The number of striations in the caudal fin is increasing. A series
of evaginations representing the gill anlagen are visible on the posterior surface of gill arches (GA). The
operculum (Op) has enlarged and covers all four gill arches.
In Figure A the dorsally pigmented swim bladder is illustrated caudal to the liver (Li). The ventricle
of the heart (HV) is large and thick-walled. Note the location and differentiation of the otic capsule, third
(3v) and fourth (4v) ventricle, meten-mesencephalic fissure (MF), and median fin fold (MFF). The
planes of section for Figures C and D are indicated by dashed lines (c) and (d), respectively (80x, LD).
In Figure B the large paired otoliths (O) are visible in the otic capsule. Stellate melanocytes (Me) are
visible on the yolk sac. The optic cups (OC) appear as heavily pigmented masses in the head region
(80x, LS). In Figure C the dorsal tube (DT) that connects the thick-walled stomach to the swim bladder
is visible ventral to the notochord (N). The gall bladder and bile duct are visible medial to the liver
tissue. The large somites (S) are bounded by the layer of epidermal ectoderm (200x, PS). In Figure D the
43
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operculum can be seen lateral to the aortic arches in the gill arches. Note the presence of olfactory
grooves medial to the optic cups and the centrally located pharynx (Ph) (lOOx, PS).
44
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II
Stage 31: Large operculum.
Plate 31
Under control conditions (at 25°C) a few fish hatch out of the chorion at this stage. Blood flow in the
aortic arches has increased. The gill anlagen have enlarged. The operculum covers the otic capsule
(OtC). The lower jaw (LJ) is well-formed. Striations are prominent in the caudal and pectoral fins (PF).
Pigment cells continue to migrate anteriorly on the ventral yolk sac (Y). The swim bladder (SB) has
increased in size and pigmentation, and maintains its attachment to the gut by a small dorsal tube. The
intestine has become pigmented. Bile production has increased, as indicated by large areas of yellow
pigment in the gall bladder and intestine.
Figure A is a line drawing that gives the overall appearance of the embryo. Note that the swim blad-
der and liver continue to enlarge. Planes of section of Figures C and D are indicated by dashed lines (c)
and (d), respectively (80x, LD). Figure B shows how the embryo has become more streamlined in
appearance due to a decrease in size of the yolk sac and a straightening of the embryonic axis. Note the
large notochord (N), the blood filled heart (H), swim bladder, and melanocytes (Me) (80x, LS). In Figure
C white matter is seen on the lateral aspects of the tectal lobes anterior to the otic capsule (200x, PS).
Figure D is a transverse serial section that shows the epiphysis (Ep) dorsal to the diencephalon. The
lateral aspects of the diencephalon are covered with white matter. Lens-shaped neuromeres (Nm) are
visible associated with the epidermal ectoderm. The triangular pharynx (Ph) is also visible in this section
(200x, PS).
45
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32
Stage 32: Gill anlagen, hatching. Plate 32
The majority of the embryos have hatched out of the chorion at this stage. The operculum and lower
jaw (LJ) are well-formed and move often. The superior, lateral, and medial rectus muscles are visible in
serial sections and result in frequent eye movement. The yolk sac (Y) is much reduced and has become
more streamlined in shape. A strong blood flow is present through the gill anlagen. The lumen of the
stomach has enlarged, and thickenings in its walls are visible histologically in both anterior and posterior
regions. Blood flow in the liver sinusoids (LS) has increased. A large neuromast is visible laterally on
each operculum. The pronephric ducts are adjacent to the cardinal veins on their lateral ventral surface,
although no glomeruli are present.
Figure A is a composite line drawing that illustrates the reduced yolk sac, as well as the medial fin
fold (MFF), melanocytes (Me), cloaca (Cl), somites (S), swim bladder (SB), liver (Li), pectoral fins
(PF). gill arches (GA), otoliths (O) in the otic capsule (OtC), heart (H), choroid fissure (CF), lens (L)
and lower jaw. Plane of section of Figure C is indicated by the dashed line (80x, LD). In Figure B the
notochord is visible extending up to the posterior edge of the distinctive optic cup. Stellate melanocytes
continue to migrate over the yolk sac (80x, LS). Figure C is a transverse serial section that shows the gall
bladder (GB) and the dorsal tube (DT) that connects the foregut (Fg) with the swim bladder. The liver
sinusoids appear as clear spaces in the liver tissue. Note the presence of the spinal cord (SC) and the
lateral somites (S) (200x, PS). Figure D is a sagittal serial section that shows the large otic capsule and
white matter (WM) of the mesencephalon (Me), as well as the ventral choroid fissure in the optic cup
(200x.PS).
46
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