208 VITAL STAINING AND MORPHOGENETIC MOVEMENTS 



A second method of staining the grey crescent is to utilize the jelly of the egg by 

 spreading it gently onto a small square of filter paper just enough to hold the egg 

 firmly in place. Take a stiff piece of dry and colored agar pellet held with forceps 

 and, using it as a pencil, mark the region of the grey crescent by applying and hold- 

 ing the dye against the egg for as long a period as possible. (Do not overstain. ) 



Study the movement of the stained grey crescent, and determine its relation to the 

 first cleavage furrow and to the subsequent position of the initial involution of gastru- 

 lation. 



2. Blastula Stage: At about the 64-128 cell stage (stage #8 Rana) apply 4 stain marks of 

 two colors around the germ ring, alternating the colors. These spots should appear 

 circumferentially placed. On other blastulae of similar stage, apply a line of alter- 

 nating colors from the germ ring of one side, through the dorsal hemisphere, to the 

 germ ring of the other side. 



These stained areas should not only move but should change shape, depending upon 

 their location in relation to the morphogenetic movements of gastrulation. (See 

 Goerttler, 1925 and Vogt, 1925.) 



3. Gastrula Stage: At the first indication of gastrulation (stage #10) mark the dorsal, the 

 lateral, and the (presumptive) ventral lip regions of the future blastopore. If you are 

 successful in this work, attempt to repeat Goerttler' s work of staining a line of spots 

 both dorsal and ventral to the initial involution of the blastopore. (See Hamburger '60) 



4. Yolk Plug Stage : (stage #11 Rana or Amblystoma) 



a. Presumptive notochord: Carefully apply a stain to the medium upper lip of the 

 early blastopore. When this embryo has reached the tail-bud stage (Rana, stage 

 #17 or #18) dissect it with needles to locate the position of the invaginated colored 

 cells. 



b. Presumptive medullary plate: Locate a region about 2/3 of the distance from the 

 dorsal lip of the early blastopore to the center of the animal pole, and mark with 

 Neutral Red. This region should not invaginate and may be followed until it be- 

 comes enclosed in the neural folds. 



5. The Somite Area of the Blastula : Using the map of Vogt (derived by the vital dye 

 method) locate the presumptive somite area (dor so-lateral to the crecentric blasto- 

 pore) and stain (on either side) with Neutral Red. When the embryo reaches the tail- 

 bud stage (stage #17 or #18) dissect it with needles and a hair loop to expose the 

 somites beneath the dorso-lateral ectoderm. 



6. Presumptive Eye-forming Areas: This can be accomplished best by using stage #13 

 of Amblystoma and consulting the exercise on Eye Field Operations to determine the 

 presumptive area to be stained. With Urodele material either Nile blue sulphate or 

 Neutral Red may be used, since there is less pigment than in the Anural egg. 



The presumptive eye-forming areas will be incorporated within the brain to give rise 

 to the optic vesicles. The stain must be applied anteriorly over the transverse neural 

 folds. Embryos thus stained should be dissected at stages #20, #26, and #30 to lo- 

 cate the position of the previously stained areas. 



7. Lateral Line Organs : Select specimens of Amblystoma punctatum at stage #30, when 

 the otic (auditory) vesicles begin to form. There may be some muscular activity 

 making it necessary to confine the embryo more rigidly, or even to cover it with a 

 piece of cover glass during the staining. Locate the otic vesicles, just above the 

 second visceral arch. Apply the dye to the otocyst and to the epidermis just posterior 

 to it for about 30 minutes. 



The lateral line organs may be followed superficially from their point of origin into 

 the tail, hence this part of the exercise requires a series of drawings to indicate the 



