Normal Amphibian Development

• The African clawed frog, Xenopus laevis (Figure 5.2) is one of the big 6 model genetic organisms in which animal development is studied.
• The stages of embryonic development in Xenopus are well known and appear as a stage series.
• There are 5 major phases of development. Each of these phases contains a varying number of stages that can be identified by one or more external characteristic.
• The 5 major phases are cleavage, blastula, gastrulation, neurulation and the tail bud phase.
• We will first look at these phases, some selected stages, and become familiar with the terminology by using a series of slides showing a stage series for development in Xenopus.
• After you become familiar with what is occurring at each stage you will watch a series of videos to observe Xenopus larvae as they develop.

Normal Amphibian Development     

• Notice in Stage 1 that the zygote (a fertilized egg following the fusion of the sperm and egg) is divided into 2 poles or hemispheres. The upper animal pole and the lower vegetal pole. The egg orientates this way due to gravity. The heavy, yolk rich vegetal pole will always be on the bottom.
• The upper animal pole is the point where the polar bodies are found. The entire animal hemisphere is pigmented with melanin (black pigment) in most amphibian species to protect the developing embryo from UV radiation and help camouflage it.
• There is a large amount of yolk concentrated at the lower vegetal hemisphere.
• The eggs are classified mesolecithal (pronounced mesa-less-i-full). This means they contain moderate amounts of yolk.  
•  In eggs that have just been fertilized you will see a lightly pigmented gray crescent-shaped region marking directly opposite the point of sperm entry (Stage 1). It is formed by a shifting of the highly pigmented cortical cytoplasm relative to the less pigmented cytoplasm beneath it. The Gray crescent material is the precursor to the notochordal mesoderm.
• The first phase of development following fertilization is Cleavage. In this phase the egg, or portions of the egg, (depending on the organism) divide by mitosis into a number of cells. These divisions are rapid and during this time the embryo does not increase in size.
•  In amphibians the first cleavage usually bisects the gray crescent (Stage 2)
•  The first, second and fourth cleavage furrows start are the animal pole and work their way toward the vegetal pole (Stages 2, 3 & 5).
•   The third and fifth cleavage furrows are perpendicular to these (Stages 4 & 6).
•  Notice in the mid-cleavage stages, the cells in vegetal half are larger and less numerous than those in the animal half. This is due to the yolk concentrating in vegetal hemisphere.  The yolk slows down cleavage in the vegetal region and by mid-cleavage the animal half has pulled way ahead in its cleavage rate and has more, but smaller cells.
• Following cleavage the embryo enters a short phase called Blastula phase (Stage 9). The embryo becomes a hollow ball of cells called a blastula. The cavity that forms inside the blastula is called a blastocoel.
• Notice that the embryo looks smoother and less 'knobby" as the cells form a tighter ball.
• After the Blastula phase the embryo enters Gastrulation. During gastrulation cells move and rearrange themselves into 3 germ layers - an outer ectoderm, a middle mesoderm and and an inner endoderm.
• If the embryo has just started gastrulation (Stage 10) you will see a small indentation called the blastopore. This is the point at which cells are moving inward to form the archenteron (literally the ancient gut. It is the primitive gut formed during this phase).
• In deuterstomes (like amphibians and mammals) the blastopore becomes the anus.
• In protostomes (like insects and worms) the blastopore becomes the mouth.
• In amphibians, surface cells move toward blastopore and inward first at dorsal lip of the blastopore, then over the lateral lips and finally the ventral lip of blastopore in process called involution.
• Once the entire blastopore is formed, a small plug of yolky endodermal cells, called the yolk plug, is caught between lips of blastopore (Stage 11).
• The yolk plug gets smaller (Stage 12) and is eventually internalized.
• Following gastrulation movements, the next phase, Neurulation, starts. During this stage the cells of the neural endoderm move for form a neural plates that fuse to form the neural tube.
• Notice the small ridge of neural ectoderm that rises above the embryo to form the neural plate (Stage 13).
• The neural plates become the neural folds as they begin to fold inward towards each other (Stage 15).
•  The sides of the neural folds gradually meet in midline and fuse to form the neural tube (Stage 17).
•  Around this time embryos start rotating within the vitelline envelop as they become ciliated. This occurs just after the two sides of neural plate have met at the midline.
• At this point the brain begins to look subdivided and budges of the optic cups are obvious (Stage 20).
• The optic cup is the precursor to the neural and pigmented retinas of the eye. During development, optic vesicles that form as outpocketings of the brain invaginate to form the optic cups.
• The embryo now enters the Tail Bud phase. As the name suggest a tail bud, which will eventually form the tail, begins to form (Stage 22)
• The body also elongates during this phase.
• By Stage 29 a gill plate will form, which eventually will be molded into external gills.
• Between Stages 37-38 pigmented cells appear and spread over the tail. The eye, mouth and gills become more obvious during this stage.
• Anurans have secondary gills enclosed in an opercular chamber. This closes them off except for single (sometimes 2) opercular opening(s).
• If embryos are ready to hatch they begin secreting a hatching enzyme that degraded the vitelline envelop and allows them to escape.
•  When hatching is about to occur the embryos begin contracting their musculature, which look like sudden twitches movements and the heart will start beating.
•  At hatching Anuran and Urodele larvae look very similar but soon begin to look different.
•  Urodeles (salamander) larvae are carnivores. Their body continues to elongate, and their gills become long and a pair of feather-like balancers develop. These act as an aid in keeping larva from sinking.
•  Anuran larvae (tadpoles) have a body that is ovoid with a long narrow tail. The intestines are especially noticeable as  long and coiled in short body.
• Anurans are vegetarians, lack balancers, but have an oral sucker that allows them to stick to objects.

Environmental Conditions Affecting Amphibian Development

•  Declines in amphibian populations have been reported over wide geographic areas including in the Americas, Britain, Australia, and the Amazon Basin.
• The decline appears to be due mostly to loss of wetlands, increased UV radiation, and pollution of breeding sites.
•  Amphibians are especially vulnerable to habitat changes because most develop in water. The eggs and embryos are only surrounded by permeable jelly coat and a vitelline membrane leaving them unprotected from pollutants.
• Many different environmental factors can adversely effect amphibian development.
• One is acid precipitation (rain & snow). This lowers the pH of breeding sites. The pH levels of temporary pools are more significantly affected than those of larger permanent pools.
• It is estimated that 10-27% of temporary ponds in the northeast have pH levels less than 5.
•  Since temporary pools provide breeding sites for~30% of all salamanders and ~50% of all frog species in North America, their acidity could have significant effects on populations.
• Sensitivity to acid varies widely among species.
•  Embryonic stages of some species show complete mortality at pH levels 4-5. This is a typical range for acidified pools. Some embryos, but not many, will survive at this range.
• Acidity has been shown to slow larval growth in many species that it doesn’t kill.
•  This extends period required for larvae to complete metamorphosis leaving them vulnerable for longer.
•  Lower pH levels cause aluminum to leach out of the substrate. Higher levels of aluminum have been found to be very toxic to early larval stages.
•  Another threat is long stretches of dry weather which dry up temporary pools, killing the embryos and larvae
• Sudden changes in temperature are also a threat. Amphibians usually have a wide range of temperature tolerance (Rana sylvatia can withstand a range from 5-21 C˚) but a sudden freeze can kill off embryos near water surface.
•  Worst is the synergistic effects of acid stress, temperature and aluminum.
•  Another hazard for amphibian development is with the thinning of the ozone layer allowing more UV radiation to reach the earths surface. UV radiation is particularly damaging to amphibians because of long exposure of their embryos to sunlight.
• During this lab we will be measuring the effects of pH levels on amphibian development.