Historical roots of centrosome research: discovery of Boveri's microscope slides in Würzburg

Abstract

Boveri's visionary monograph 'Ueber die Natur der Centrosomen' (On the nature of centrosomes) in 1900 was founded primarily on microscopic observations of cleaving eggs of sea urchins and the roundworm parasite Ascaris. As Boveri wrote in the introductory paragraph, his interests were less about morphological aspects of centrosomes, but rather aimed at an understanding of their physiological role during cell division. The remarkable transition from observations of tiny dot-like structures in fixed and sectioned material to a unified theory of centrosome function (which in essence still holds true today) cannot be fully appreciated without examining Boveri's starting material, the histological specimens. It was generally assumed that the microscope slides were lost during the bombing of the Zoological Institute in Würzburg at the end of WWII. Here, I describe the discovery of a number of Boveri's original microscope slides with serial sections of early sea urchin and Ascaris embryos, stained by Heidenhain's iron haematoxylin method. Some slides bear handwritten notes and sketches by Boveri. Evidence is presented that the newly discovered slides are part of the original material used by Boveri for his seminal centrosome monograph.

Keywords: Ascaris and sea urchin eggs; Theodor Boveri; University of Würzburg; centrioles; centrosomes; cleavage division.

Figure 1.

Theodor Boveri at the age of 29 in Munich (1891). At that time, Edmund B. Wilson spent some months at the Zoological Institute in Munich to work with Boveri. Wilson about Boveri: he was ‘far more than a brilliant scientific discoverer and teacher. He was a manysided man, gifted in many directions, an excellent musician, a good amateur painter, and we found many points of contact far outside of the realm of science. The best that he gave me was at the Café Heck where we used to dine together, drinking wonderful Bavarian beer, playing billiards, and talking endlessly about all manner of things’ [9]. Courtesy of the archive of the University of Würzburg.

Figure 2.

Folders with microscope slides used by Boveri for his centrosome studies. The slides can be dated to the period from 1894 to about 1898. Each slide contains several rows of serial sections of sea urchin and Ascaris embryos at the first cleavage stages, stained with Heidenhain's haematoxylin. Boveri's handwritten notes are on the outside of the folders (a) and on several slides with sea urchin (b) or Ascaris material (c,d; the roundish objects on the slides are oviduct cross sections). In addition, specific sections are marked by Indian ink dots or their position is indicated (d; II.7–9 stands for second row, sections 7–9).

Figure 3.

(a) In order to facilitate histological processing of sea urchin eggs, batches of fixed eggs were wrapped by Boveri in shed pieces of salamander skin (an epidermal cell nucleus is denoted by the arrow) and processed like a piece of tissue. (b) Even after prolonged destaining (differentiation) of iron haematoxylin-stained sections of Ascaris oviduct, the contact sites of the tightly packed eggs retain the stain. The resulting staining pattern is diagnostic for the haematoxylin method. (c) The same situation depicted by Boveri (adapted from [7]). Scale bars, 50 µm.

Figure 4.

Images of centrosomes and centrioles during the first cleavage division of Echinus microtuberculatus eggs. Comparison between photographs taken from original slides (a–d) and Boveri's drawings (a′–d′, adapted from [7]). Centrosomes were described by Boveri as moderately and uniformly stained spheres (a,a′) or as spheres interspersed with a filamentous scaffold (b,b′). Astral rays appear to radiate out from the centrosomal surface. Occasionally, a tiny dot is recognized in the centre of a destained centrosome, most probably a centriole (insert in b, arrow). (c) This micrograph of overstained, yet clearly delineated centrosomes was taken from a slide labelled ‘Sobatta II. s. stark’ (Sobotta II, very strong). Boveri wrote [, p. 33] that he intentionally left some slides for 8 days in the staining solution leading to totally black centrosomes (c′). (d) Two tiny dots, apparently connected by fine filaments (‘Verbindungsstrang’), are seen inside an anaphase centrosome (arrows). A comparable situation is depicted in the drawing (d′). Scale bars, (a–d) 20 µm and (insert in b) 10 µm.

Figure 5.

Flattening, expansion and division of centrosomes in preparation for the second cleavage of Echinus microtuberculatus eggs. Comparison between photographs taken from original slides (a–c, all from the Wheeler series) and Boveri's drawings (a′–c′, adapted from [7]). (a,a′) At telophase of the first division (shown is the karyomere stage when individual chromosomes become surrounded by a nuclear envelope), centrosomes no longer appear as compact spheres but are spread out in the form of disc-like structures. (b,b′) Upon fusion of the karyomeres the centrosomal discs attach to the poleward faces of the daughter nuclei. (c,c′) Eventually each elongated centrosome divides, the two halves condense into compact structures and migrate to opposite positions of the daughter nuclei to form the new spindle poles. Scale bars, 20 µm.

Figure 6.

(a) Microscope folder with Boveri's handwritten note ’Eier von A. meg., nicht aufgeklebte Schnitte. Färbg. nach Delafield‘ (Eggs of Ascaris meg., sections not affixed. Staining according to Delafield). The paraffin sections were applied to glass slides without using adhesives such as gelatin or egg albumen which might have interfered with the staining procedure. (b) Photograph of a section through a dividing Ascaris egg, stained with Delafield's haemalum. Note the absence of centrosome staining. Astral rays terminate at ill-defined focal bodies within each spindle pole (arrows). The reddish tinge results from en bloc staining of the eggs with borax carmine prior to sectioning. The arrowhead denotes a polar body. Scale bar, 20 µm.

Figure 7.

(a) Centrosomes and (b) centrioles at metaphase of the zygotic division of Ascaris eggs and duplication of the centrosome in a postmitotic daughter cell (c). The corresponding drawings by Boveri are adapted from [7] and are shown below the photographs. (a) The centrosomes are uniformly stained and appear as relatively large, black spheres (arrows). Upon overstaining, they do not grow further but the proximal regions of the astral fibres radiating from the sharply demarcated centrosome body become progressively stained (insert in a). (b) Upon extensive destaining (differentiation) of the sections, a minute dot appears in each centrosome, the centriole (arrows). (c) An early phase of centrosome duplication in one of the blastomeres, prior to migration of the sister centrosomes (arrows) to opposite poles of the nucleus (the blastomere on the right-hand side is only partially included in the section). (a–c) Scale bars, 20 µm and (insert in a) 10 µm.

Figure 8.

A rare example of a sea urchin egg cleaving by a tripolar spindle, found in one of the slides. A triaster will divide simultaneously into three blastomeres with random assortments from the chromosomes seen as dark objects in the centre of the image. Boveri realized the importance of such abnormal divisions as a means to study the effect of differing chromosome complements on development. In 1902 he wrote that multipolar mitoses might lead to the development of tumour-like formations [23]. Scale bar, 20 µm. (Online version in colour.)