Differentiation (1984) 26: 176-181 Differentiation CQ Springer-Verlag 1984

Discovery and initial characterization of a new conditional (temperature-sensitive) maternal effect mutation in the axolotl Robert Briggs' and Francoise Briggs Program in Molecular, Cellular, and Developmental Biology, Department of Biology, lndiana University. Bloomington, IN 47405, USA

Abstract. The discovery of a new temperature-sensitive ma- ternal effect mutation in Ambystoma mexicanum is de- scribed. The new gene (ts-1) was recognized when 100% of the eggs spawned by homozygous females failed to devel- op past early gastrulation when reared at 25" C. Eggs raised at 10" C developed normally to sexual maturity. A tempera- ture-sensitive period during blastulation was identified by a preliminary series of temperature shifts. Histologic exami- nation revealed that nuclear abnormalities, especially chro- mosomal bridges, characterized ts-1 embryos reared beyond the ninth cleavage at the restrictive temperature (25" C).

Introduction A major portion of the developmental program for early postfertilization in amphibian embryogenesis is built into the egg during oogenesis [reviewed in 41. The large number of genes which are active in oogenesis contribute informa- tional, structural, and metabolic components -which are deployed following activation of the egg by sperm penetra- tion. In order to gain insight into the manner in which those cytoplasmic components control early pattern specifi- cation, Humphrey actively pursued the identification of ax- olotl maternal effect mutations. Several were recognized and have been studied by various investigators [reviewed in 2, 7,8]. In each instance females which were homozygous for the maternal effect gene spawned eggs which - regard- less of the genotype of the fertilizing sperm - arrested prior to the completion of organogenesis.

Since the amphibian embryo is especially suited for em- bryologic analyses, the search for additional axolotl mater- nal effect genes has continued. The present report describes the discovery and preliminary characterization of a new conditional (temperature-sensitive) maternal effect muta- tion.

Methods

The mutant gene described in this report was discovered in the Tompkins/Delanney line. Female #4198.1 (in which the gene was initially discovered) was related to a male which was imported from Mexico in 1968. That male is in the lineage of all ts-1 females subsequently recognized, so it might have been the source of the mutation. This

1 Deceased

new gene was assigned the name 'temperature sensitive-I ' (ts-1).

All breeding animals are routinely maintained at 18"-20" C and eggs from spawnings were reared using stan- dard methods for amphibian embryos. Eggs were routinely staged according to the number of cleavage divisions they had completed at the time they were manipulated. For histologic examination eggs were fixed in San Felici's [6], embedded, sectioned (10 pm), and stained with Feulgen and fast green.

Discovery of Is-1

This new mutation was discovered in 1979 when a spawning (# 4811) generated by the Indiana University Axolotl Col- ony exhibited abnormal patterns of early embryogenesis. In this spawning all embryos maintained at room tempera- ture (20-22" C) exhibited major developmental defects, par- ticularly during gastrulation. The set of embryos main- tained at 10" C, however, developed normally, became feed- ing larvae, and attained sexual maturity. All subsequent spawnings from the same female (# 4198.1) were tested at 25", 18", and 10" C. The developmental stage of onset of the defects and their severity depended on the temperature at which the embryos were reared. At 25" C the embryos arrested just before or during gastrulation. Occasionally one or more embryos reached the tailbud stage but displayed defects caused by incomplete or defective gastrulation. Em- bryos raised at the intermediate temperature (18" C) fre- quently manifested a wide range of defects including arrest at late blastula, incomplete gastrulation, swollen gut at the tailbud stage, or inability to establish normal circulation. Figure 1 demonstrates several abnormal morphologic char- acteristics.

Genetic basis of the ts-1 phenotype

Female #4198.1 provided the first spawning of tempera- ture-sensitive eggs. In repeated (total = 6) spawnings of that female the temperature-sensitive phenotype was equally ex- pressed, regardless of the genotype of the male (Table 1). Two female cousins of # 4198.1 were test mated. They pro- duced eggs which exhibited the same temperature sensitivity originally observed for 4198.1 eggs (Table 2).

Of the six spawnings from female # 4198.1, # 5057 (a backcross) and # 4967 (an outcross) provided important information on the genetic basis of ts-1 when progeny test-

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Table 1. Repeated spawnings of femalc # 4198.1 produced ternpera- ture-sensitive eggs

Spawn- Date Male # Relationship Characteristics of male of spawning to 4198.1

ing #

481 1 8/3/79 4222.7 Unrelated Room temperature- arrest (primarily at gastrulation); 10" C-survival

4838 2/5/79 3794.8 Related 25' C arrest at gastrulation

4871 1/9/79 C-7 Unrelated 25' C arrest at gastrulation

4967 25/1/80 4746.5 Unrelated 25' C arrest a t gastrulation

5057 13/5/80 4838.2 Son of 4198.1 25" C arrest at (backcross) gastrulation

5153 26/12/80 4894.1 Unrelated 25" C arrest at gastrulation

ing was carried out. Of the eight #4967 females which spawned, none gave temperature-sensitive embryos. This result suggests that ts-1 is not a dominant gene. However, one of the five #SO57 backcross females so tested gave embryos which displayed the ts-1 syndrome exactly as de- scribed above. Continued progeny testing showed similar results, leading to the conclusion that ts-1 represents a new recessive, conditional, maternal-effect gene. All progeny testing has so far shown no obvious linkage to the other well characterized genes (c-cardiac; t-twisted gill ; h-hand; d-white) present in stocks of the Tompkins/Delanney line.

The temperature-sensitive phenotype associated with ts- 1 was compared with another temperature-sensitive pheno- type - vasodilation (gene v) - discovered earlier by Briggs and Humphrey [3]. A comparison of the two phenotypes is provided in Table 3. The ts-1 mutant differs from the u mutant in several ways. Most notably, the u phenotype is highly variable, even at the restrictive temperature (25" C). The proportion of the embryos which exhibit the various effects varies, as does the stage of onset of the effects. The cytologic features of the ts-1 phenotype (see below) at the restrictive temperature are unique, and unlike those associated with the gene v.

Results

Experimental analysis of the temperature sensitive period

The temperature-sensitive period was analyzed by perform- ing a preliminary series of temperature shifts on developing embryos. At 30-60 min intervals eggs were collected and transferred to 20% Holtfreter solution equilibrated at either 10" or 25" C. The 'shiftdown' eggs were transferred to 10" C at hourly intervals from the onset of cleavage. At

Fig. 1 a-c. External morphology of embryos spawned by ts-llts-1 female. a and b Embryos raised at 25" C arrest at gastrulation or, occasionally, a t tailbud stage; c typical characteristics of ar- rested embryos raised at intermediate temperatures (18-22" c)

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Table 2. Temperature sensitivity of eggs spawned by cousins of female # 4198.1

Female # Spawning # Date Temperature Terminal stage

Total # Gastru- Neural Hatching Feeding eggs lation fold larvae to

adult

- - 4301.4 5065 24/5/80 25" C 41 34 7 20" c 209 94 115 18" C 51 - 48 3 10" c 43 - 6 - 37

7-2-2 5033 9/4/80 25" C 29 16 7 3 20" c 33 13 3 17 10" c 16 1 - 1 14

- - -

- -

Table 3. Comparison of two temperature sensitive phenotypes

Temperature Embryos from v/v females Embryos from ts-l/ts-1 females

25" C 6 9 0 % arrested at range of stages from early cleavage (stages 5-6) through gastrulation. Majority of early arrested embryos display the zoned or platinum appearance peculiar to embryos from v/v females About 6 8 0 % loss of embryos during gastrulation, neurulation. Stratification of pigment definite but not as pronounced as in 25" C set

15-20" C

10" c

Cytology at 25" C

Somc arrest of embryos (2&60%) at gastrulation or neurulation. Some survive to adulthood Stratification of cytoplasmic components, cessation of mitosis, very large nuclei. Concen- tration of RNA in upper region of cells

90-100% embryos arrested at stage 8-9. Early cleavage embryos normal through stage 7, i.e., no pigment stratification, etc., (Fig. 3)

Complete range of defects, including arrest a t stage 8, exogastrulation effects, 'swollen gut' tailbuds, and inadequate circulation in hatchlings. Less than 20% survived at 20" C 8&90% survival to adulthood if embryos are kept at 10" C through stage 9 Anaphase bridges evident in pre-arrest embryos at stage 7-8. No stratification of pigment or other cytoplasmic components

Table 4. Preliminary characterization of the temperature-sensitive period by temperature shifts

Direction Cleav- Total Extent of development

temperature at time em- Blas- Gas- Ab- Feeding shift of bryos tula trula normal larvae

of age# #

shift post- neurula

25"+1OoC 3 12 1 11 6 11 1 10 9 14 2 12

12 14 14 10"+25"C 3 10 10

6 9 9 9 12 3 9

12 5 4 1

25' C each hour beyond first cleavage corresponded closely to successive cleavage divisions. The ' shift-up' eggs were carefully observed and at each cleavage division several eggs were transferred to 25" C. Control sets of embryos were maintained throughout embryogenesis at either 10' or 25" C.

The results of the experiment are tabulated in Table 4 and summarized in Fig. 2. From the shift-down procedure it is apparent that a temperature-sensitive period exists dur- ing the interval between the 9th and 12th cleavages ( 6 1 3 h

k 25' $ 10'

3 6 9 1 2

25' lo' 1

10'- I

0 5 25'8

10'- c I K E 25'

10' I I I b e 1 3 6 9 1 2

Cleavage # Fig. 2. Summary of temperature shift data included in Table 4. Top. Shift-up indicates sensitive period occurs between 9th and 12th cleavages. Borrom. Shift-down indicates that by 12th cleavage Is-1 eggs cannot be rescued by low temperature

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Table 5. Nuclear cytology of ts-f embryos

Cleav- #em- Non-mitotic Anaphases and telophases age # bryos nuclei

ex- Normal With Percent amined bridges with

bridges

ts-f Embryos reared at 25" C 2 3 Normal 3 2 Normal 4 1 Normal 5 - 6 4 Normal 6-7 3 Normal 9 4 Normal

12 3 Abnormal 13 3 Abnormal 14-15 6 Abnormal

ts-1 Embryos reared at 10" C 2 2 Normal 4 2 Normal 7 2 Normal

13 2 Normal

Normal embryos reared at 25" C 9 Normal 9 Normal

13-14 Normal 13-14 Normal 13-14 Normal 13-14 Normal 1>14 Normal 13-14 Normal

13 0 0% 14 66 83% 21 15 42% 90 20 18%

65 3?

11 0 16 0 21 0 20 0 32 l ? 37 0 33 0 36 0

at 25" c). The Sbft-UP data suggest that by the 9th cleavage the temperature-sensitive period is almost past. The mid- blastula stages (100-500 cells) appear, therefore, to be the most sensitive to the restrictive temperature.

Fig. 3a, b. 1s-f embryos (25" C) prior to developmental arrest. a Ex- ternal morphology of cleaving embryo; b normal chromosomal cy- tology at ninth cleavage

Descriptive studies - Cytology of ts-1 embryos reared at the restrictive temperoture

At various stages embryos were fixed, stained, and exam- ined for histologic abnormalities. The data are included in Table 5 and briefly summarized below:

Cleavage stage. Several embryos were examined at early cleavage stages. No abnormalities in either cytoplasmic or nuclear components were detected (Fig. 3).

Blastulation. A variety of abnormalities, each associated with the nucleus, were observed. Most prominently, several nuclei exhibited anaphase or telophase bridges which indi- cated either non-disjunction or incomplete replication (or both). In fact, at around the 12th cleavage most telophases contained bridged chromosomes (Fig. 4a). Mitotic figures also occasionally exhibited variable chromosome numbers,

phase were Often in size and form- Some were large and contained dispersed chromatin while others were rounded-up, small, and pycnotic (Fig. 4b). Small round cells were sometimes located in the blastocoel.

chromosome and acentric chromosomes. Inter- Fig. 4a-d. Nuclear cytology of ts-1 embryos. a Twelfth cleavage cells display telophase bridges; b round cells of blastula with pyc- notic interphase nuclei; c 1415th cleavage cells exhibit telophase with clumped chromosomes; d 15th cleavage cells with chrorno- somes in telophase bridges

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Gastrulation. Gastrulae displayed the cytologic abnormali- ties mentioned above (Figs. 4c and d). Occasionally, non- nucleated cells were observed.

Luter stages. Examination of a 'swollen gut' tailbud re- vealed an enlarged archenteron which contained several en- dodermal cells with pycnotic nuclei. Late tailbud stage em- bryos were usually somewhat microcephalic. Although they should have contained blood cells in the heart, gills, and pronephric sinuses none were observed. It appears, there- fore, that IS-1 embryos develop normally through the first eight or nine cleavages. Then various chromosomal aberra- tions can be detected. Those aberrations persist through the tailbud stages to developmental arrest. The occurrence of those chromosomal abnormalities coincides with the temperature-sensitive period (Fig. 2).

Discussion

Conditional maternal effect mutants of the type described in this report (Fig. 5) are particularly useful for studies in experimental embryology. On the one hand, the conditional (temperature-sensitive) nature of the phenotype facilitates the identification and rearing of the homozygous females. By raising larvae in the presence of estradiol it has been possible to increase the yield of females from any given spawning [l]. That procedure is currently in use in our laboratory and promises to facilitate the raising of breeding stocks of maternal effects such as ts-I. On the other hand, conditional mutants which arrest (under restrictive condi- tions) at a relatively early stage in embryogenesis are poten- tially valuable for analyzing the mechanisms which regulate specific events. In the case of ts-2, arrest occurs during the cleavage stages when a transition from synchronous to asynchronous cell division occurs [lo] as a consequence of variable G1 phases [S] . Preliminary experiments reveal that the temperature sensitive period occurs at around the time of the 9th-12th cleavage. Newport and Kirschner [9] recently demonstrated that a mid-blastula developmental transition occurs at just about this stage (stage 12) in Xeno- pus embryos. At that stage, activation of RNA transcription is initiated. The rs-1 mutation might prove valuable for further analyzing that transition. Further experiments are planned to define more precisely the temperature-sensitive period. Although a single major temperature period was identified (Fig. 2), perhaps other less pronounced tempera- ture sensitivie periods also exist.

During the temperature-sensitive period distinct cyto- logic (nuclear) abnormalities were observed. The majority of anaphases and telophases seen in sections of embryos which developed through the 9th-12th cleavages at the re- strictive temperature displayed chromosome bridges. Those bridges probably lead to chromosome breaks and rearran- gements which in turn lead to the aneuploidy often observed during gastrulation. That aneuploidy may eventually gener- ate the developmental arrest syndromes which characterize the mutant phenotype (Fig. 1). Perhaps the endoderm cells are more severely affected than other cells. Resulting mal- functions of archenteron cells might generate the 'swollen gut' syndrome seen at intermediate temperatures (e.g., 18-20' C). At least three models can be formulated to ac- count for the temperature-sensitive phenotype: 1) 'Stage-specijk 'model. A critical event which requires the product of the normal allele of ts-1 occurs during the inter-

-+ 10. 0

(0- Fig. 5. Diagrammatic illustration of the is-1 mutant phenotype

2000

1000

400

100

50

20

Anaphare Bridges land Dthu IUCIOQ obnormoll?loa)

6 7 e s 10 ii 12 13 14 is 16 ir ie is

Fig. 6. Relationship of nuclear abnormalities to cleavage division. The chromosomal abnormalities are first detected only after the transition from synchronous to asynchronous cleavage divisions (arrow). Graph redrawn from data in Reference [lo]

Cleavage #

Val between the 9th and 12th cleavages. For example, the product of the mutant ts-1 gene might be a temperature- sensitive enzyme which is required to regulate mitosis dur- ing that developmental stage. Eggs spawned by ts-llts-1 females, containing abnormal (temperature-sensitive) en- zyme arrest when exposed to 25" C during the period when the function of that enzyme is first required (9th-12th cleav- ages). The data on the temperature shifts (Fig. 2) are consis- tent with this model. The anaphase bridges and other cyto- logic defects observed at the 9th-12th cleavages are also easily explained by that model. Curiously, the time of ap- pearance of the nuclear abnormalities coincides with the transition from a synchronous to an asynchronous cleavage rate (Fig. 6). Perhaps an enzyme which is involved in the control of that transition is affected by ts-1.

2) 'Low temperature rescue ' model. Structural components produced during oogenesis by the normal allele of the ts-1 gene self-assemble and function (passively) during early em- bryogenesis. For example, cytoskeletal elements involved in karyokinesis probably are required for orderly cleavage. During early cleavage at the higher temperature (25" C) structural imbalances due to abnormal ts-1 gene products can be accomodated, since the number of new cells formed per unit of time is relatively low. Eventually, however, when the number of new cells formed per unit of time increases dramatically (e.g., 9th-12th cleavages) at 25" C the abnor-

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ma1 gene product rate-limits karyokinesis and anaphase bridges result. Developmental arrest follows. At the lower temperature (loo C) the function of the abnormal structural components outpaces the requirements of karyokinesis until either the normal allele of rs-1 contributed by the sperm nucleus becomes active, or the regulation of karyokinesis changes to become independent of the ts-1 gene product. The temperature shift data as well as the data on anaphase bridges are consistent with this model. Evidence to support this model would be forthcoming if future analyses reveal that eggs spawned by ts-1 females are in one way or another defective at relatively early (e.g., 1 st-9th cleavages) stages of embryogenesis.

A third, perhaps more speculative model can also be formulated, based upon recent data from Signoret's labora- tory. During early cleavage a heavy DNA ligase (8s) rep- laces a light (6s) form [ll]. At low temperature (10OC) no replacement occurs, yet development proceeds normally. The 8s form is therefore dispensable. However, at elevated temperatures (18" C) the 8s form does appear (J. Signoret, personal communication). Such a replacement of a temper- ature-sensitive non-allelic enzyme could also be postulated to explain the ts-1 phenotype. For example, at the permis- sive temperature such a phenomenon may also occur in ts-1 eggs. The rescue could be accomplished by the expres- sion of a replacement enzyme.

Acknowledgements. This investigation was supported by PHS grant # GM 29307. Dot Barone provided excellent technical assistance, and George M. Malacinski provided F.B. with invaluable help in the writing of the manuscript and preparation of the illustra- tions.

References 1. Bodney SA (1982) Towards thc developmcnt of a practical

method for reversing the sex (male to femalc) of large numbers of Ambystoma mexicanum. Axolotl Newsletter 11 : 12-18

2. Briggs R (1 973) Developmental genetics of the axolotl. In: Rud- dle FH (ed) Genetic mechanisms of development. Academic Press, New York, pp 169-199

3. Briggs R, Humphrey RR (1962) Studies on the maternal effect of the semi-lethal gene (0) in the Mexican axolotl. I. Influence of temperature on the expression of the effect. IT. Cytological changes in the affected embryos. Dev Biol 5:127-146

4. Davidson EH (1976) Gene activity in early development. Aca- demic Press, New York

5. Graham CF, Morgan RW (1966) Changes in cell cycle during early amphibian development. Dev Biol 1 4 : 4 3 9 4

6. Humason GL (1972) Animal tissue techniques. Freeman, San Francisco

7. Humphrey RR (1975) The axolotl, Ambystoma mexicanum. In: King RC (ed) Handbook of genetics, Vol4. Plenum Press, New York, pp 3-17

8. Malacinski GM (1978) The Mexican axolotl, Ambystoma mexi- canum: Its biology and developmental genetics, and its autono- mous cell-lethal genes. Am Zoo1 18 : 195-206

9. Newport J, Kirschner M (1982) A major developmental transi- tion in early Xenopus embryos. 11. Control of the onset of tran- scription. Cell 30: 687-696

10. Signoret J, Lefrense J (1971) Contribution a I'etude de la seg- mentation de l'oeuf d'axolotl. I. Definition de la transition blas- tuleenne. Ann Embryo1 Morphog 4: 113-123

11. Signoret J, David J-C, Lefresne J, Houillon C (1983) Control of DNA ligase molecular forms in nucleocytoplasmic combina- tions of axolotl and Pleurodeles. Proc Natl Acad Sci USA 80:3368-3371

Received August 1983 / Accepted in revised form November 1983