Centrosomes Fail to Attach to Pronuclear Envelopes in fue Embryos (A–D) In vitro fertilized embryos from WT and fue females fixed and labeled for centrosomes (γ-tubulin antibody, red) and DNA (DAPI, blue) at 10 (A and C) and 15 mpf (B and D). Asterisks indicate polar bodies, and male and female pronuclei are indicated with symbols. Scale bar represents 20 μm and applies to all panels. Images are projections from confocal z stacks. (E and F) Distance between centrosomal γ-tubulin labeling and male or female pronuclear envelopes quantified at 10 and 15 mpf. Error bars indicate ±1 SE. See also Figure S1 and Movies S1 and S2.

lrmp Transcripts Show Dynamic Localization Patterns Lost in fue Mutants (A) Chromogenic in situ hybridization with lrmp antisense probes in WT (left) and fue mutants (center) and negative control sense probes (right). (B) WT embryos fixed at 5 min intervals and labeled with γ-tubulin antibody (red) and DAPI (blue), in combination with fluorescent in situ detection of lrmp mRNA (green). Scale bar represents 20 μm and applies to all panels in (B). See also Figure S3.

Lrmp Protein Localizes to Nuclear Membranes and Subregions of the Mitotic Apparatus WT embryos fixed at 5 min intervals and labeled with γ-tubulin antibody (red), anti-LrmpMD antiserum (green), and DAPI (blue). Scale bars represent 20 μm. See also Figure S4.

C-Terminal Domains of Lrmp Facilitate Subcellular Targeting (A) Diagram of EFGP-fusion constructs. (B) Fusion construct RNAs encoding EGFP::LrmpCC-TM-L (top row), EGFP::LrmpTM-L (second row), and EGFP::LrmpCC construct (bottom two rows) were injected into one-cell WT embryos. Embryos expressing EGFP were fixed and processed for DAPI and anti-γ-tubulin immunostaining between 2.5 and 3.5 hpf. White boxes indicate fields shown at higher magnification (right). Scale bar represents 20 μm in all lower magnification panels. (C) Summary of results from EGFP::Lrmp C-terminal protein expression. Low viability (far right column) manifested as cell division defects and failure to undergo gastrulation, which led to embryo lysis. See also Figure S5.

Mutant Oocyte Injection and Rescue with WT lrmp mRNA Stage IV oocytes from fue mutant females were isolated and injected with WT lrmp mRNA. Following maturation and in vitro fertilization, embryos were fixed at 1 hpf and labeled for DNA, γ-tubulin, and Lrmp. Examples of weakly rescued embryos (top row), moderately rescued embryos (second row), and strongly rescued embryos (third and fourth rows) are shown. White box in the third row indicates the region shown at higher magnification in the fourth row. Embryos from uninjected fue oocytes, derived from the same set of mothers and treated in parallel with injected oocytes, showed the typical mutant phenotype (bottom row). Scale bars represent 20 μm. See also Table S2.

fue Embryos Fail to Undergo Pronuclear Migration and Fusion although the Sperm Aster Is Intact, Related to Figure 1

(A) DAPI (blue) and β-catenin (green) immunolabeling of wild-type embryos at the 4-cell stage.
(B) Same labeling as (A) in fue embryos shows that male and female pronuclei do not fuse though cytokinetic divisions proceed normally resulting in anucleate blastomeres. Scale bar in (A) represents 75 μm and applies to (A,B).
(C-F) Immunolabeling of embryos with anti-α-tubulin antibody to visualize the sperm aster. The sperm aster is of similar size and morphology in both wild-type and fue embryos at 7 and 10 mpf. Arrowheads indicate residual meiotic spindle microtubules while arrows point to the sperm aster. Scale bar in (C) represents 20 μm and applies to (C-F).

Western Blotting and Additional Protein/RNA Localization Analysis, Related to Figure 4

(A) Embryo lysates from wild-type and two fue mutant clutches at cleavage stages and 4 hpf were probed with anti-LrmpMD antibody. A band at 200 kDa (red arrow) likely corresponds to Lrmp and appeared reduced in mutant compared to wild-type, while a shorter 160 kDa band was present in mutant lysates. The observed 200 kDa molecular weight is greater than predicted for the zebrafish Lrmp isoforms (approximately 159 kDa - 162 kDa), however mouse and human Lrmp proteins also exhibit higher-than-expected molecular weights on denaturing gels (predicted at 62 kDa and 59 kDa, observed at 75 kDa and 69 kDa, respectively) [11,44]. The identity of the 25 kDa band seen in both mutant and wild-type is not known and unlike the 200 kDa species, was not recognized by the more C-terminal Lrmp antibody (data not shown). Wild-type 24 hpf lysates did not show any prominent signal. Anti-actin antibody was used as a loading control.
(B) fue embryos were labeled with DAPI (blue), γ-tubulin antibody (red) and anti-LrmpMD antibody (green). Localized Lrmp protein was undetectable in the majority of imaged fue embryos (16 out of 20), and only very faint nuclear envelope labeling could be seen in the remaining mutants (4 of 20).
(C) In unfertilized activated wild-type eggs at 15 minutes post-activation (mpa), Lrmp protein is localized at the female pronuclear membrane at levels comparable to wild-type zygotes (top row; compare to Figure 4, top three rows). Similar to fertilized fue zygotes, localized Lrmp protein is not readily detectable in unfertilized fue eggs (bottom row).
(D) Fluorescent in situ labeling against lrmp mRNA (green) in combination with antibody labeling for Lrmp protein (red) in wild-type embryos. In early mitosis, Lrmp protein and lrmp mRNA are offset with Lrmp protein closer to the condensing DNA (top row). During chromosome segregation at anaphase, lrmp mRNA and Lrmp protein colocalize in spindle regions but only Lrmp protein appears at the reforming nuclear membranes (second row). Lrmp protein alone continues to localize at nuclear membranes in late mitosis, when Lrmp protein labeling at centrosomes again becomes pronounced, co-localizing with lrmp mRNA in this structure (bottom row in (D)). In (B,C), male and female pronuclei are indicated with symbols while polar bodies are denoted by white asterisks. Scale bars represent 20 μm.

Additional Observations on Fusion Protein Localization and Alignment of the Lrmp C-Terminal Domain to Known KASH Proteins, Related to Figure 5

(A-B) Embryos injected with fusion construct RNAs at the 1-cell stage were fixed at 2.5-3.5 hpf, then labeled with DAPI and anti-γ-tubulin antibody.
(A) Wild-type embryos injected with EGFP::LrmpCC-TM show nuclear membrane and spindle localization of EGFP as well as EGFP aggregation, similar to what is seen with the EGFP::LrmpCC-TM-L fusions.
(B) Localization of overexpressed fusion constructs in fue mutant embryos. Fusion proteins showed subcellular localization patterns similar to those observed when overexpressed in wild-type embryos, namely nuclear membrane localization of the EGFP::LrmpTM-L fusion construct (top row), and centrosomal plus cell membrane localization of the EGFP::LrmpCC-TM construct (bottom row). Scale bars represent 20 μm and apply to all panels in A,B. White boxes indicate regions shown at higher magnification in right-most panels.
(C) Diagram summarizing the roles of the different Lrmp C-terminal subdomains in protein localization.
(D) ClustalW was used to align the C-terminal 60 residues of zebrafish and human LRMP and several KASH proteins from various species. Invariant residues are indicated with an asterisk below the alignment while positions with conserved similarity are marked with a colon (substitutions with highly similar properties) or a period (substitutions with weakly similar properties).