The molecular embryology of the brine shrimp

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TIBS - May 1976

REVIEWS The molecular embryology of the brine shrimp C h r i s t o p h e r C. H e n t s c h e l a n d J. R. T a t a

The availability o f unlimited amounts o f desiccated, ametabolic, yet viable gastrulae has caused the brine shrimp, Artemia salina, to become increasingly popular as a subject for developmental biochemists.

Fig. I. Development of the brine slwimp Artemia

salina.

Young adult Artemia.

Molecular embryologists have tended to be rather conservative in the number of systems which have attracted more than their mere cursory scrutiny. The preeminence of such organisms as the sea urchin or the South African clawed toad Xenopus laevis are not without good reason since they each afford peculiar advantages to the experimenter. Nevertheless, the concentration of effort on a few organisms brings with it the danger of prematurely indicating consistent results as general principles when, in fact, they are only one of a number of possibilities. Thus, the central dogma of modern embryology that development proceeds by the spatially co-ordinated synthesis of new cell types,

mediated by variable gene expression, does not appear to be an accurate description of the early development of the brine shrimp Artemia salina, The biology ofArtemia salina is unusual in a number of ways. It has often been compared to plants, since strains exist of various ploidities some of which are bisexual, some parthenogenetic. There is a world-wide distribution of these strains in saline or alkaline lakes to which habitat they have evolved some remarkable adaptations. A comprehensive bibliography of early literature on the subject (over 400 papers are described) has been compiled [1]. As one of the adaptations to their unNational Institute for Medical Research, Mill Hill, usual habitat development may proceed by one of two routes (Fig. 1); either London, NWt, U.K.

development continues right up to the swimming nauplius stage (Fig. 2c) in the female ovisac, or in conditions of extreme salinity with low oxygen tension, such as occur when salines start drying up, development is interrupted at the gastrula stage and a seed-like cyst is formed which is shed into the brine. Desiccated Artemia cysts (Fig. 2a) are gathered in commercial quantities due to the excellence of the nauplii as food for fish farmers or for pet aquaria. The ability to buy the desiccated, ametabolic yet viable cysts in unlimited quantities and to reinitiate development merely by placing them in aerated salt solutions has made them favourite objects for experimentation in toxicology, radiobiology and developmental biology. A crucial early observation [2] in cytological studies of the post-gastrular development of ,4rtemia cysts indicatecl that no new cell synthesis was taking place up to the pre-nauplius stage (Fig. 2b) since the number of nuclei per embryo remained statistically constant at about 4000. However, considerable internal differentiation has taken place - perhaps most obviously the formation of the eyespot - though the abundance of yolk makes this difficult to see. Moreover, even the first naupliar stage

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shows only 10% more nuclei than the gastrula. This uncoupling of new cell synthesis from differentiation and morphogenesis has been confirmed by studies of the DNA content [20] and extended by the use of inhibitors of DNA synthesis at the naupliar stage. If newly hatched nauplii are treated with 5 fluorodeoxyuridine [3], DNA synthesis is effectively blocked, but the larvae continue to elongate and indeed are identical to controls for about two days. Protein synthesis

(a)

(b)

rc)

Fig 2. Major developmental stages in Artemia. (a) Desiccated Artemia O'sts. (b) Pre-naapli~ stage showing some pear-shaped embryos fidiy emerged from O'StS and others in the process of emerging. (c) Swimmh~g naupli~ stage.

The remarkable separation of morphogenesis from new cell synthesis in the initial post-gastrular development of A r t e m i a makes it an ideal system for assessing the role of protein synthesis in executing the developmental programme, since any changes in biochemical parameters clearly reflect differentiation of pre-existing cells. Early studies by Clegg et al. [4,5] and Hultin et al. [6]indicated that the protein synthetic apparatus of the dormant embryos and the changes taking place on the resumption of development were strikingly similar to events taking place on the resumption of development of seeds or newly fertilized eggs. Thus, the dormant A r t e m i a gastrulae contain a large store of unprogrammed and metabolically inactive monosomes with little or no polysomal material being detectable. On the resumption of development, however, polysome formation is demonstrable within minutes and protein synthesis is re-initiated. During the first four hours of development the embryos retain the ability to be desiccated without loss of viability. If development is restarted a second time the initial events are repeated. This apparent reversibility of early developmental events was investigated by Morris [7] who concluded that development from the gastrula to the nauplius was bi-phasic with an initial reversible preparatory phase preceding a non-reversible phase of active morphogenesis and differentiation. The cytological events defining the transition between the reversible and non-reversible components remain unclear. If development is allowed to proceed uninterrupted for some hours a generalized increase in the workings and efficiency of the protein synthetic apparatus occurs. The biochemical mechanisms underlying these changes appear to be highly complex with modifications of nearly all the constituents having been reported. Thus, physico-chemical changes in the ribosomes occur [8], elongation factor EF~ changes from the aggregate heavy form (EFzH) to the light form (EF~L) [9] and changes in the transfer RNA [10] and amino acid populations are detected [1 l].

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Particularly interesting have been the findings by Ochoa's group [12,21] that the dormant cysts are deficient in a number of initiation factors, and that these are gradually replenished during the first few hours of development. The deficiency of factors in the dormant Artemia embryos contrasts with other dormant embryos, such as the wheat-germ, which have all the factors required for protein synthesis. However, like the wheat-germ embryo the Artemia embryo is a useful source of ribosomes for cell-free protein synthesis, there being practically no background in the absence of added messenger RNA, the missing factors being derived from either activated embryos or from a heterologous source. The origin and intracellular distribution of messenger RNA populations directing early protein synthesis has been investigated by Hultin et al. [13,14]. Here again, the analogy with events taking place in seeds and unfertilized eggs appears to hold. In these systems the very rapid resumption of protein synthesis, as well as its insensitivity to actinomycin D, an inhibitor of transcription, led to the suggestion that early protein synthesis utilized pre-existing 'masked' forms of messenger RNA. The criteria for establishing the existence of such 'masked' messengers in dormant Artemia cysts has been largely fulfilled. Thus, the demonstration of the recovery from dormant embryos of membrane associated poly(A)-containing heterogeneous RNA which is an active template in a cell-free system as well as the presence of ribonucleoprotein particles containing poly(A)-containing RNA which when deproteinized are also active templates [15], leaves little doubt that these are 'masked' messages. Unfortunately, Artemia cysts appear to be impermeable to actinomycin D prior to hatching and the inhibitor experiments cannot be performed. Scant information exists as yet on the identity of the major proteins coded for during Artemia pre-emergence development or the mode of activation of the masked messengers. Ocha's group [16] have recently tested the possibility that the 'masked' messengers are not translated because they lack the GpppG" 5'-terminus or 'cap' which appears, in cell-free systems at least, to be essential for translation. However, this did not appear to be the case since not only were at least some of the messengers from the dormant cysts 'capped' but they contained significant amounts of messenger RNA methylase activity which converts the capped end to the activated methylated form. These results do not exclude the possibility that compartmentation within the embryos

99 allows selective 'capping' of groups of masked messengers with their sequential activation. Whatever the mechanism for activating masked messengers, it seems probable that the engagement of particular groups of messengers is dependent on the developmental state since the characteristic poly(A)-containing ribonucleoprotein particles are present even after significant development. Moreover, in vitro translation products directed by Artemia polysomes show distinct developmental specificity. Inducible haemoglobins One of the few well characterized groups of proteins in Artemia are the haemoglobins [17]. Normally various forms of globin chains are synthesized during larval development with the earliest forms being detectable in polysomes even before hatching [22]. However, the rate of synthesis of globins appears to be extremely sensitive to oxygen tension of the environment. When oxygen tension is low, such as is found in conditions of very high salinity, up to a twenty-fold increase in the haemoglobin content occurs making the shrimps appear red. Oxygenation of the medium causes the levels to fall back to a base level. As mentioned previously, low oxygen tension also tends to lead to cyst production rather than viviporous reproduction. In fact, there is a good correlation between the haemoglobin levels and the switch to predominant formation of cysts with the excess haem pigments forming part of the characteristic chorion of the cyst. Although the molecular details of this process are lacking, this appears to be a remarkable example of a complex biolo,gical decision being mediated by the environment interacting with the genome. RNA synthesis The transcriptional events and changes taking place in the nuclei during reactivation and pre-emergence development in Arternia remain far less well understood than the corresponding cytoplasmic events. As in the case of protein synthesis, however, the lack of new cells during this period should allow a clearer interpretation of the role of transcription. The lack of progress results from two technical problems: the low permeability of the cysts to direct precursors of RNA synthesis and the difficulty of purifying nuclei from the large numbers ofco-sedimenting yolk platelets [18]. Nevertheless, the use of t4CO2 as a precursor of RNA synthesis, via its incorporation into pyrimidine nucleotides [4], has indicated that the initiation of RNA synthesis occurs within fifteen minutes of the resumption of development, although the rate of synthesis at this

stage is very low. Notwithstanding the technical difficulties of using ~4C02 as an RNA precursor (protein is also heavily labelled), the earliest RNA species synthesized appeared to be ribosomal RNA despite the large surplus of ribosomes in the dormant embryos. This is a situation common at the gastrular stage of many embryos. Recently, and in contradiction to earlier reports, Susheela [19] has found that the dormant cysts can be made substantially permeable to 32p if they are dechorionated with hypochloride - a procedure which does not harm the viability of the embryo. Using this procedure a high rate of labelling of AMP pools was detected at early time points but no synthesis of heavy forms of RNA could be demonstrated until emergence. Hopefully, the discrepancies between these two views of early RNA synthesis will soon be resolved. Newly labelled nauplii are in contrast to pre-emergent embryos highly permeable and have proved to be more amenable to biochemical analysis. RNA synthesis is very intense at this stage [20] with all species being heavily labelled by 32p or 3Hadenosine. In experimental conditions a peak rate of synthesis is followed by a rapid drop to undetectable levels, despite an increase in the specific activity of the precursor pool. Unfortunately, in the work cited, as in much of the biochemical work on larval development, the Artemia nauplii are in fact starving and in excessively crowded conditions. Under optimal conditions the nauplii are voracious feeders capable of achieving a 500-fold increase of biomass in two weeks, whereas under experimental conditions they survive off yolk stores for a number of days while suffering drastic decreases in weight and calorific value despite continuation of elongation and morphogenesis. Thus, the finding that there is a steep drop in the post-hatching rate of RNA synthesis may simply reflect the conditions of starvation. Relevant to this point is the study by Bagshaw et al. [23] who investigated the levels ofsolubilized RNA polymerase during this period. Substantial levels of polymerases I and II were detected shortly after hatching, but after two days in culture without food a drastic reduction of the level of the ribosomal RNA synthesizing polymeruse I was observed; a result typical of tissues during starvation. DNA synthesis The conspicuous absence of DNA synthesis from the gastrular stage up to the pre-nauplius poses intriguing questions as to the mechanisms controlling the onset of DNA synthesis in Artemia embryos. One suggestion [3] has been that a close

I00 link exists between the metabolism o f an. unusual dinucleotide, diguanosine tetraphosphate, and the initiation o f D N A synthesis. The presence o f large amounts of diguanosine tetraphosphate (and the related triphosphate) in Artemia cysts, but not in adult shrimps, indicated that they might play some key role in early embryonic development. Two main conclusions have emerged from studies o f the metabolism o f these nucleotides during development. First, that diguanosine polyphosphates are the sole source of purines during early development until dietary purine is ingested at the larval stages. Artemia appears to have no de novo purine synthetic pathway and has a specific and absolute requirement for dietary purine in order to reach maturity. Secondly, the pool of dATP serving as a precursor to initial D N A synthesis after hatching is compartmentalized and derives almost exclusively from diguanosine tetraphosphate even when dietary adenine supplies the acid-soluble pool. The pathway o f this tight coupling between diguanosine tetraphosphate metabolism and D N A synthesis is unknown but presumably involves initial splitting of the dinucleotide to GTP and G M P (an asymmetric pyrophosphohydrolase catalyzing this reaction has been characterized), followed by conversion of guanine to adenine and ribose reduction. The physical location o f the guanosine polyphosphates in the yolk platelets [18] suggests that these ubiquitous organelles, far from acting as passive food stores, are ultimately linked with the execution o f the developmental programme. The importance of substances originating from yolk platelets in exerting control over the genome during development has been described in a number o f organisms and may be an aspect o f the cytoplasmic dominance o f nuclear events. Cryptobiosis Finally, although the focus o f this review has centred around the synthesis o f macromolecules during the resumption o f post-gastrular development from dormant cysts, it should be pointed out that the cysts themselves are a remarkable biological material worthy o f further study. Artemia cysts are one o f the best characterized cryptobiotic materials; a term denoting a state in which metabolism has come reversibly to a standstill through desiccation and characterized by incredible resistance to environmental stress. Artemia cysts have, for example, been kept close to absolute zero for six days without loss of viability. A result taken to indicate that all the information required for life even in these complex embryos resides solely in their structure. The biochemical and

TIBS - May 1976

biophysical basis o f these properties are largely unknown but challenge the commonly held view that many macromolecular assemblies, particularly those largely formed by hydrophobic interactions, such as membrane systems, suffer irreversible breakdown when removed from an aqueous milieu.

References 1 Littlepage,J. L. and McGinley,M.N. (1965) San Francisco Soc. Spec. PubL, No. I 2 Nakanishi, Y.H., lwasaki, T., Okigaki, T. and Kato, H. (1962) Annot. Zool. Jap. 35, 223-228 3 Finamore, F.J. and Clegg,J. S. (1969) in The Cell Cycle (Padilla, G.M., Whitson, G.L. and Cameron, I. L., eds),p. 249, AcademicPress, New York 4 Clegg,J.S. and Gulub, A. L. (1969) Develop. Biol. 19, 178-200 5 Gulub, A. L. and Clegg, J. S. (1968) Develop. Biol.

17, 644-656 6 Hultin, T. and Morris, J. E. (1968) Develop. Biol. 17, 143-164 7 Morris,J. E. (I 971) Comp. Biochem. Physiol. 39A, 843-85'7 8 Clauwaert, J. (1974) Arch. Int. Physiol. Bioehim. 82 (5), 972-973 9 Slobin, L.I. and Mollen. W. (1975) Nature 258, 452-454

10 Bagshaw,J.C., Finamore, F.J. and Novelli, G.O. (1970) Develop. Biol. 23, 23_-25 11 Emerson, O.N. (1967) Comp. Bioehem. PhysioL 20, 245-261 12 Sierra,J. M., Meier,O. and Ochoa, S. (1974) Proc. Nat. Acad. Sei. USA 68, 1331-1335 13 Nilson, M. O. and H ultin, T. (1974) Develop. Biol. 38, 138-149 14 Nilson, M.O. and Hultin, T. (1975) FEBS Left. 52, 269-272 15 Grosfeld, H. and Littauer, U.Z. (1975) Biochem. Biophys. Res. Commun. 67, 176-182 16 Muthukrishnan, S., Filipowiez,W., Sierra, J.M., Both, G.W., Shalkin, A.J. and Ochoa, S. (1975) J. BioL Chem. 250, 9336-9341 17 Bowen, S.T., Lebhera, H.G., Poon, M., Chow, V.H.S. and Grigliatte, T.A. (1969) Comp. Biochem. PhysioL 31,737-747 18 Warner, A. H., Poudzioukas,S. G. and Finamore, F.J. (1972) Exp. Cell Res. 78, 365-375 19 Susheela,C. and Jayaraman, K. (1976) Differentiation 5, 29-33 20 McClean, O.K. and Warner, A.H. (1971) Develop. Biol. 24, 88-105 21 Filipowicz,W., Sierra, J. M. and Ochoa, S. (1975) Proc. Nat. Aead. Sci. USA 72, 3947-3951 22 Moens, L. and Kondo, M. (1975) FEBS IOth Congr. Abstr. 508 23 Birndorf, H.C., D'Alessio.J. and Bagshaw,J.C. (1975) Develop. Biol. 45, 34-43

Biochemical transportation of genetic information Kenneth Murray Restriction endonucleases provide new opportunities for the specific breakage o f D N A into large fragments that can be rejoined in any order. D N A sequences from any source may thus be inserted into plasmids or phage and cloned in bacterial cells.

Genetic engineering is by no means new, but the recent surge o f interest in the topic arises from the new found ability to transpose by biochemical methods large segments o f DNA from one source to another, by-passing the natural barriers for transfer o f genetic material between different species. These exercises began with experiments in which D N A molecules from two different strains o f eoliphage 3. were broken by mechanical shearing and the half molecules from each strain separated. The successive use o f 2 exonuclease and polynu~cleotide ligase then permitted the left halves o f one population o f D N A molecules to be joined to the right halves o f the other to give a recombinant D N A preparation from which viable phage could be recovered by transformation ofEscheriehia eoli spheroplasts [1]. This sequence o f reactions depends upon a degree o f overlap K.M. is Reader in Molecular Biology, University of Edinburgh, Scotland, U.K.

in sequences o f the D N A fragments in the population o f halves produced by shearing, so that complementary sequences exposed by the action o f the exonuclease may be annealed and the resulting molecules trimmed and repaired in the subsequent reactions. A more generally applicable procedure was used in the construction o f hybrid D N A molecules from the 2dv gal plasmid and simian virus 40 (SV40) D N A [2]. Here two circular D N A molecules were converted into linear molecules by the action o f an enzyme that introduced a single break into each D N A and the 3' termini o f the linear molecules were extended by the addition o f several dA residues to one population o f molecules and several dT residues to the other in reactions catalyzed by polynucleotide terminal transferase. The single-stranded complementary sequences attached to the two D N A preparations could then be annealed and the molecules trimmed and repaired through the combined action o f D N A polymerase