Characterization of lamin mutation phenotypes in Drosophila and comparison to human laminopathies

Abstract

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.

Figure 1. Comparison of lamin genes from different organisms.

(A) Schematic diagram of a general lamin protein showing the central rod and IF-tail domains used in sequence comparisons. (B) Condensed cladograms showing the evolutionary relationship of 28 lamin genes, compared for the central rod domain, the IF-tail domain and full protein sequences. Protostome and deuterostome sequences group together rather than with different deuterostomic lamin groups. Note also how the central rod domain of the single C elegans lamin gene is equally related to deuterostome and protosome sequences, whereas its IF-tail domain groups with other protostomal sequences. The full C. elegans lamin sequence also occupies an intermediary position. Arrows indicate the root node of respective C.elegans sequences. Bootstrap values (1000 trials) are given as percent figures near nodes. See Materials and Methods for lamin designations, and Supplemental Figure S1 for full cladograms. Sequence alignments upon which these were based are available on request.

Figure 2. Molecular characterization of null allele lam ^D395.

(A) Schematic drawing of the lam locus. Exons are presented as large grey arrows, and relevant restriction sites and the translation start site are indicated. The site of insertion of the P element responsible for the lam ^P mutation is shown, and the bar underneath indicates the extent of the lam ^D395excision. (B) Southern blot of genomic DNA from the genotypes indicated above each lane. The white arrowheads mark the band corresponding to the second exon lacking in lam ^D395 homozygotes and lam ^D395/Df (2L) cl-h1 transheterozygotes. The white asterisks mark the 4.8 kb HinDIII band resulting from the wild type chromosome (lower) and the band from the mutant chromosome (upper), which is larger due to the deletion of the HinDIII (2910) site.

Figure 3. Adult behavior of lam mutants.

(A) Righting reflex plotted on a log scale. Each symbol represents the mean of six trials for an individual adult, with error bars showing SEM. (B) Negative geotaxis was plotted as a function of genotype and age. Each point represents 20 – 35 adults, with error bars showing SEM. Data from males and females were pooled. (C) The percentage of living adults that climbed in the negative geotaxis assay, plotted as a factor of age. Homozygous lam ^G262 mutants all died before three weeks of age.

Figure 4. Larval behavior of lam mutants.

(A) Vials showing the difference in pupariation height between wild type at left, and lam ^G262at right. (B) Chart of pupariation height in mm. Error bars correspond to SEM. Differences between lam ^D395 and lam ^D395/Df(2L)cl-h1, and outcrossing are described in the text. Sample sizes; wt (w ¹¹¹⁸) = 163, G262/+(lam ^G262/+) = 114, G262 (lam ^G262) = 133, 04643/+(lam ⁰⁴⁶⁴³/+) = 40, 04643 (lam ⁰⁴⁶⁴³ ) = 45, P/+(lam ^P/+) = 105, P (lam ^P) = 5, D395/+(lam ^D395/+) = 74, D395 (lam ^D395) = 33, G262 outcrossed = 39, 04643 outcrossed = 19, P outcrossed = 94, and D395/Df = 87. The asterisk marks lam ^P laboratory strain, where we had to screen a very large number of larvae to find 5 pupae to measure - see also the footnote to lam ^P laboratory strain lethality in Table 1. (C) Larval locomotion is shown for the indicated genotypes as the number of 5 mm squares crossed in 5 minutes (see Materials and Methods). Error bars correspond to SEM. In all cases, n = 30.

Figure 5. Muscle histology.

(A–C) Adult indirect flight muscle morphology. Thoraces were cleared and examined by polarized light microscopy. (A) Wild type w1118. (B) lamG262 and (C) lam04643. Note that this technique does not allow fine focus, but permits gross assessment of muscle bulk and organization through entire thoraces. (D–F) Abdominal segmental muscle fibers labeled by rhodamine-conjugated phalloidin. (D) Wild type w1118. Muscle 5 is indicated by a white arrow. (E–F). lam04643 showing absence (E) or misinsertion (F) of muscle 5. (G) Higher magnification of phalloidin-stained w1118 larval body wall muscles showing regular patterns. (F) lam04643 - fine structure defects are highlighted by arrowheads. Similar defects are seen in other alleles (data not shown). (I) Phalloidin-stained late stage lam04643 embryo demonstrating normal muscle organization and form.