Tumor suppressor Lzap regulates cell cycle progression, doming, and zebrafish epiboly

Abstract

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here, we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract, and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly--the earliest morphogenetic movement in animal development--which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation.

Fig. 1. lzap is Highly Conserved During Evolution

A: Gene structure of zebrafish lzap located on chromosome 12. Filled boxes represent exons. Cen, centromere; Tel, telomere. B: Alignment of human, mouse and Zebrafish Lzap protein sequences. Amino acid sequences of Lzap orthologs from the NCBI database were aligned and similarity assigned based on the Clustal W algorithm. Hs, Homo sapiens (NP_788276.1); Mm, Mus musculus (NP_084524.1); Dr, Danio rerio (NP_001002105.1). (✽) identical; (⚫) conserved; (:) semi-conserved. C: Pairwise comparison of amino acid conservation between human (H. sapiens), mouse (M. musculus), and zebrafish (D. rerio) Lzap.

Fig. 2. lzap is Expressed during Epiboly and Organogenesis

A: Relative expression of lzap mRNA in developing fish embryos. lzap expression was determined by qRT-PCR at indicated developmental stages and normalized to β-actin expression. hpf, hours post-fertilization; dpf, days post-fertilization. B–K: Representative photographs of lzap expression during development as determined by in situ hybridization. Embryos are depicted in lateral view, except for the animal pole view shown in F’. Ubiquitous expression of maternal lzap was detected throughout cleavage stages (B, C) and expression was maintained into early epiboly (D). After epiboly from 9–12 hpf, lzap expression was concentrated in the precordal plate and areas corresponding to the future hatching gland (E–F’). At 1 dpf to 2 dpf lzap expression was more intense in the CNS, eye, and pharyngeal region (G, H). lzap staining was visible in the developing pharynx region, pharyngeal arches and in the primordial of the gastrointestinal organs at 3 dpf (I). lzap mRNA was detected at 4–5 dpf (J, K) in the pancreas and gastrointestinal tube, jaw and pharynx region, tectum, gills (see inset, K) and the hindbrain regions. L: Negative control with sense probe at 3 hpf reveals no staining.

Fig. 3. Morpholino-mediated Depletion of Lzap Results in Cleavage Stage and Epiboly Defects

A: Schematic representation of lzap RNAs. lzap:3HA contains the 5’ UTR sequence targeted by the lzap morpholino (MO) and was used to confirm MO effectiveness. lzap:CDs contains the entire lzap coding sequence but lacks the MO target sequence and was used for rescue. B: Verification of on-target and dose dependent effects of lzap MO on exogenous Lzap expression. Embryos were injected with lzap:3HA singly or with increasing amounts of lzap MO visualized by immunofluorescence 4-hours post fertilization (hpf). No immunofluorescence was observed in control embryos lacking the primary antibody (data not shown). C: Schematic showing incidence of developmental defects and non-viable embryos at indicated time points following injection of lzap MO, control MO or in embryos without injection. D–E”: lzap specific MO or control was injected and viability and morphology determined by microscopic visualization at indicated times. Results were compared to uninjected embryos. D–H: Lateral view of uninjected embryos from 3–24 hpf. D’–H’, D”–E”: Lateral view of lzap MO injected embryos. Embryos were time matched to uninjected embryos. Arrows indicate persistence of the syncytial layer. Mild and severe phenotypes following injection of lzap MO are shown.

Fig. 4. Cleavage Stage and Epiboly Defects in lzap Morphants are Rescued by Co-injection of lzap mRNA

A–F: Co-injection of lzap mRNA with 5’ UTR morpholino (MO) partially rescued developmental defects of lzap morphants at 6- and 24-hours post fertilization (hpf). G: Schematic showing embryo viability at indicated time points following injection of lzap MO with or without lzap RNA and in embryos without injection. Data represents means and standard errors derived from more than 180 embryos from 3 independent experiments. H: lzap morphants display proliferative, mitotic and apoptotic defects that are partially rescued through co-injection of lzap mRNA. Embryos were analyzed at 6 hpf by immunohistochemical staining using antibodies to PCNA, p-Histone H3 and cleaved Caspase-3 as indicated (magnification, ×40). I–L: Indices for proliferation (I), mitosis (J) and apoptosis (K) were determined by dividing the number of cells staining for PCNA, p-Histone H3, or cleaved Caspase-3, respectively, by the total number of cells. Means and standard error are presented. Data is from sections of 20 independently injected embryos. For relative cell size determination, cell borders were marked on captured hematoxylin stained images and relative area (L) calculated by ImageJ software. Values are normalized to control cells. NS, not significant.

Fig. 5. Depletion of Lzap causes delay in cell cycle progression but does not disrupt zygotic gene expression after MBT

A: The expression of bmp2b, eve1, ntl and hgg1 in control and both age-matched and stage-matched lzap morphant embryos were detected by whole-mount in situ hybridization. bmp2b expression, lateral view, 6 hpf in control. eve1 and ntl expression, top view, 6 hpf in control. hgg1 expression, lateral view, 12 hpf in control. B: Cell cycle analyses of dissociated zebrafish embryonic cells. DNA content was determine on untreated embryonic cells (a), on cells treated with 10 μg/ml nocodazole for 30 min (b), or on cells treated with nocodazole followed by release for 15 (c) or 30 min (d). Cells were assigned to G1, S, or G2/M based on fluorescent signal and manual gating using CellQuest Pro software. Results in the table represent the mean of at least three independent experiments. C: Embryos with and without injection of 4ng lzap MO were co-immunostained with anti-α-tubulin (red) and p-Histone H3 (green) antibodies at 4, 6 and 9 hpf in mitotic cells. Results are representative of observations from more than 100 nuclei for each panel.