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08 March 2019

How Embryologists See: Anne McLaren’s Mouse Models

This post forms part of a series on our Science blog highlighting some of the British Library’s science collections as part of British Science Week 2019.

What does an embryo look like? You’ve probably seen pictures –photos of clumps of tiny little cells, most likely taken of a petri dish in a lab. But embryologists face many barriers when bringing these miniscule cells into vision. The developmental biologist Dr Anne McLaren found ways around some of these problems starting with her work in the 1950s.   

In 1952, the mammalian developmental biologist Dr Anne McLaren moved to UCL to begin conducting a series of experiments intended to transplant mouse embryos from the uterus of one mother to the uterus of another, foster, mother – a technique called embryo transfer. There were several reasons for her wanting to do this, but the central one was a problem of vision. She wanted to make the embryos visible. As she explained in 1960,

Experimental embryology in mammals starts with a grave and obvious disadvantage compared to experimental embryology in, say, frogs or sea-urchins - namely the relative inaccessibility of the mammalian embryo. On the other hand it is a subject of particular interest, not only because man himself, and most of his domesticated animals, are mammals, but also because the mammalian embryo goes through almost all the critical stages of development in the most intimate contact with a genetically different organism, its mother.

This intimate relationship between the embryo and its mother in the very early stages of implantation, and the potential applicability of these insights to other mammals, like humans, made this an important area of study. This relationship also represented a prime example of McLaren’s central research interest, namely how the gene and environment interact in development. In the mammal, the maternal uterus crucially provides the environment in which the genes have to exert their effects. This is why maternal effects on inherited characters are of particular interest to McLaren.


At school we are often taught that development looks something like this,

The stages of human embryo development from ovum to foetus.
Illustration: human fertilization and embryogenesis. With kind permission of Gaurab Karki, at www.onlinebiologynotes.com


McLaren saw things differently. Although the embryo could indeed develop into a foetus and a baby, this was only under particular circumstances, in a given environment. McLaren wanted to better understand what was required of this environment for the embryo to develop into a healthy mouse. Development could also go wrong, and it was certainly not as simple as the expression of a set of genes against a neutral backdrop. In fact, she believed that the whole concept of a gene meant fairly little without an adequate account of the environment through which they were expressed. 

 

‘This image has been removed due to expiry of the copyright licence. 'The Bucket Model and When Causes Interact,’ are from The Mirage of a Space Between Nature and Nurture, Evelyn Keller Fox, pp. 8-9, Copyright, 2010, Duke University Press. All rights reserved. Republished by permission of the copyright holder. www.dukeupress.edu



But the problem of being able to see this environment remained. Although she could not look directly inside the womb, McLaren realised that instead she could make the interactions taking place between the embryo and the uterus visible. This was made possible by a phenomenon that had been noticed with the number of lumbar vertebrae, the vertebra starting after the last rib attachment and running down to the last vertebra not sacralised, in the offspring of reciprocal crosses between two strains of mouse. In Problems of Egg Transfer in Mice (1955), she explained,

We suspect the existence of a maternal effect whenever reciprocal crosses are made between two genetically differing strains or varieties, if the progeny differs according to which strain was taken as the maternal parent, and which the paternal. …In species hybrids between the horse and the donkey, the mule, which has a horse mother and a donkey father, differs in a number of respects from the hinny, which has the donkey mother and the horse father. One difference lies in the number of lumbar vertebrae that the animals have. Most mules have 6 lumbar vertebrae, like their mothers; while most hinnies have 5 lumbar vertebrae, again like their mothers.

Another example of this effect observed in mules by John Hammond and Arthur Walton in 1938, was the case of lumbar vertebrae in mice. E. L. Green and W. L. Russel, working at Bar Harbor in New York in 1943, noticed such a phenomenon, a suspected maternal effect on lumbar vertebrae in mice, but their experiments had been stopped short by a fire in their laboratory. The effect presented McLaren with an observable trait that was definitely not just due to chromosomal sex linkage, because the difference also appeared in female progeny of the crossed strains, who of course carry two of the same X chromosome. Even through the trait was not sex-linked, it could still be determined either by the cytoplasm of the egg or the uterine environment that the mother provides. The case thus provided a specific instance of the question of the respective roles of gene and environment in the inheritance of an observable trait. The best way of distinguishing between these contributions, she decided, would be by transferring eggs between females of the two strains, “since such eggs would have the cytoplasm of one strain but the uterine environment of the other” (Research Talk, 1953). If the influence was exerted through the cytoplasm, the young would be unaltered in phenotype by the transfer; but if it was exerted through the uterine environment, the reciprocal difference would be reversed.

Sketch showing an ovum being influenced by either its genotype or the environment.
Image: Is it the uterus or the egg affecting the number of vertebrae of the mice? Copyright estate of Anne McLaren MS89202/12


Embryo transfer techniques had been around for a while – in fact, the pioneer of the technique, Walter Heape had used the technique as early as 1890, to show the exact opposite of what McLaren suspected was the case with lumbar vertebrae – namely that the uterus had absolutely no effect on the developing embryo. As their experiments progressed, McLaren and her then husband and collaborator Donald Michie showed that the uterus, in the case of lumbar vertebrae, did exert an effect on the embryo. The mice in the surrogate uterus expressed the trait of the surrogate, not the genetic mother.  There was something in the maternal uterus, not the cytoplasm, that effected the number of lumbar vertebrae. By the end of the experiment she was not able to determine exactly how  this effect was exerted but, she reflected in 1985, the message of the experiment was clear,

As to how this influence is exerted, from the physiological point of view, we are so far in complete ignorance. But the general moral for the geneticist, I think, is clear: that is, when we are dealing with mammals we must be prepared to extend our picture of the genetic control of morphogenetic processes, to envisage their regulation not only by the action of the embryo's genes, but also by the action of the genes of the maternal organism in which the embryo is gestated

Turning cauliflowers into mice: mouse model growing pains 

As might be expected with such a new technique, it took a while to perfect it, to be able to produce standardised results. In the process, McLaren began to see some unusual things. Indeed, during the early days of the experiments, McLaren and Michie were worried about the appearance of some the fertilised ova being produced by the donor female after they’d administered the hormones to induce ovulation. In a research talk from 1953, McLaren recounts,

During the Summer of last year, we were using two-day eggs only; and one day, actually the day we were rejoicing because for the first time we’d got transferred eggs to develop into mice, our 2-day eggs, instead of looking like normal mouse eggs with 4 or 8 distinct spherical blastomeres, suddenly began to look like cauliflowers. The blastomeres coalesced, and the eggs looked awful.

She goes on,

From that day onward, all their eggs looked like that, and as it seemed obvious that something looking like a cauliflower couldn’t develop into a mouse, we didn’t even bother to transplant many of them, but spent much fruitless effort trying to find the cause of the trouble. However, we’ve now got over this difficulty, partly because by using 3-day eggs, which look quite normal, as well as 2-day eggs; partly because this Summer only some of our 2-day eggs looked like cauliflowers; and partly because we’ve got some evidence that cauliflowers can in fact develop into mice.

These pages from McLaren’s lab notebooks show how she tested different variables, like the PH of the medium in the dish before transfer to the foster mother, or the daylight to which embryos were being exposed. She obtained some strange shapes in the process.

Image of written lines in a notebook
Strange cauliflower shapes. Detail from Anne McLaren’s ‘UCL Embryo Transfer’ laboratory notebook, 1953-1956. Copyright estate of Anne McLaren (Add MS 83843).
Image of written lines in a notebook
‘Ghosts’, or disappearing, eggs. Detail from Anne McLaren’s ‘UCL Embryo Transfer’ laboratory notebook, 1953-1956. Copyright estate of Anne McLaren (Add MS 83843).
Image of written lines in a notebook.
A healthy blastocyst (Cells differentiated into cell layers, preceding the embryo stage) –‘hooray’! Detail from Anne McLaren’s ‘UCL Embryo Transfer’ laboratory notebook, 1953-1956. Copyright estate of Anne McLaren (Add MS 83843).

McLaren was discovering new things about the ways in which embryos could develop, and she didn’t always understand what was going on. The appearance of these cauliflowers in development point to the limited view she was getting. It remained difficult to visualise what was going on at these early stages inside the maternal uterus, and the best the embryologist could do was to set up an limited model of the process, to bring to the fore some of the phenomena she was interested in. But biological models, unlike the ones we draw or build out of inanimate material, don’t always comply. Moreover, the view was always partial, and in this case especially limited because all she could do was move her embryos between uteri –about which she knew very little. The only way of knowing more about the uterus would be by intervening in this environment, changing it in some ways and observing the effects this had on the developing embryo which was impossible while the womb remained inaccessible.  As we shall see, McLaren soon went on to develop another window that would allow her to visualise more directly the forces acting on the embryo during development. 

From wombs to dishes

As far as her interest in making the interactions between uterus and embryo visible was concerned, McLaren had definitely succeeded. She had done this by intervening in the biological process of gestation, by moving an embryo from one mother to another and observing the effects it had on the developing embryo. As we have just seen, this technique threw up obstacles and limitations. The cauliflower effect was just one example of a malformation that McLaren was unable to explain because she had little idea about what the uterine environment was made of. She could not figure out the exact mechanisms by which the uterus acted on the embryo because, in order to do this, she would have to play around with them like she had with the medium in the dish prior to transfer, to isolate different variables until she could figure out what factors were at work. She would have to manipulate to be able to see. At the same time, however, McLaren was developing a very promising technique that could provide the solution – the technique of embryo culture. Writing in 1958, she mentioned a method by which egg transfer enables the experiment to influence the environment of the early mouse embryo directly, instead of through the medium of the mother or the other embryos. In collaboration with Dr. Biggers, I have been culturing 8-16 cell mouse embryos according to the technique of Whitten, on Krebs bicarbonate with glucose and bovine plasma albumen added. In two days at 37 [symbol: degrees], nearly 100% of such embryos reach the blastocyst stage, a development which in vivo takes only one day. I then transferred these blastocysts to the uteri of pregnant female recipients, and found that their viability relative to that of control blastocysts had been in no way impaired by the culture treatment….So far we have done no more than demonstrate the feasibility of the technique; but it seems to me that a study of the effects upon subsequent development of variation in the conditions of culture and the constitution of the culture medium, might provide yet another means to overcome the inaccessibility of the mammalian embryo…

Embryos in dishes would allow McLaren to figure out the conditions needed for normal embryonic development. When she and John Biggers (1958) later showed that a mouse embryo after being cultured outside the womb for over 24 hours, could be replaced in the uterus of a mouse mother and develop into a normal healthy mouse, they had pathed the way for In Vitro Fertilisation in humans that would become a reality 20 years later. IVF, a technique that changed the field of embryology as well as society at large, was just one of the offshoots of McLaren’s explorations of gene-environment interactions.

Marieke Bigg
Ph.D candidate, University of Cambridge

Further reading:

McLaren, Anne, and J. D. Biggers. 1958. ‘Successful Development and Birth of Mice Cultivated in Vitro as Early Embryos.’ Nature 182 (September): 877.
McLaren, Anne. 1958, 1960. Experimental studies on the effect of the prenatal environment. 
McLaren, Anne. 1985. An effect of the uterine environment. 

Marieke Bigg is a Ph.D candidate at the University of Cambridge. After completing a B.A. Honors in comparative literature at the University of Amsterdam, she obtained an M.Phil in sociology from the University of Cambridge. In her current PhD research, which she conducts under the supervision of Professor Sarah Franklin, she draws on the biography of Anne McLaren to map the debates on human embryo research in Britain in the 1980s, and proposes new models for policy-making in the area of human fertilisation and embryology today. She is funded by the Wellcome Trust.

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