Immunohistochemical analysis of vtd gene’s neurodevelopment role in Drosophila embryo
INTRODUCTION
Molecular Genetics of Disease Pathology
Cornelia De Lange Syndrome is a developmental disorder characterized by growth delays, mental disability, facial dysmorphisms, and upper limb malformation in a spectrum of severity. The genetic disorder has an incidence of 1 in 10,000-30,000 newborns (OMIM, 2013). Heterozygous genetic mutations linked to this phenotype have been studied in Drosophila orthologs and include genes Nipped-B, SMC1, SMC3, and verthandi (vtd, also known as RAD21 in humans), the responsible gene of choice for this experimental analysis (Bloomington Drosophila Stock Center).
Classical CDLS involves a defect in the NIPBL regulatory gene, responsible for about 75% of cases; the remaining incidence is attributed to mutations in either of the 3 core cohesin components SMC1A, SMC3, or RAD21—or the cohesin recycling enzyme HDAC8. Cohesin, a highly-conserved protein ring complex, holds sister chromatids together in mitotic cells, ensuring proper segregation to opposite poles during division and proliferation; the vtd gene encodes cohesin’s Rad21 protein sub-unit. Rad21 protease cleavage causes premature sister chromatid disjunction and widespread chromosome missegregation in proliferating cells, lethal in postmitotic neurons (Pauli, 2008 via Society for Developmental Biology).
Studies of the C. elegans ortholog mau-2 linked cohesin with axon guidance during development (Bernard, 2004 via SDB). Investigation of γ neurons in Drosophila suggests that cohesin mediates the elimination of axon projections and dendrites. Cleavage of vtd‘s product Rad21 abolishes the developmentally controlled pruning of axons and dendrites in γ neurons. However, as the axon-projection defects were detected in developmentally arrested late pupae, the results do not rule out the function for cohesin in regulating axon guidance, because Rad21 cleavage might be incomplete when γ-neuron axons begin growing out in the first place (Pauli, 2008).
Research suggests additional roles for cohesin and vtd in DNA double-strand break repair and regulating gene expression (Nasmyth, 2005 via SDB). Part of the trxG gene family also involved in regulating the hedgehog (hh) gene, vtd/Rad21 is integral in the cohesin ring complex’s speculated role facilitating transcriptional activation. Because of vtd’s location deep within the centric heterochromatin of the left third arm of the Drosophila chromosome, little is known of its characterization at the molecular level (Hallson, 2008 via SDB).
This experiment’s focus on neurodevelopmental vtd gene mutation in the fruit fly model, linked to the known phenotype/human developmental disorder CDLS, explores genetic function during embryonic neural development—characterizing effects on axon fasciculation (FASCILIN II expression), as well as axonogenesis and apoptosis (22c10/FUTSCH expression) in a comparison of wild-type expression patterns of target axonal proteins with those in the mutant strain.
FASCILIN II (FAS II) protein is normally expressed in three axonal connectives in the CNS, as well as peripherally. FAS II is involved in protein binding and axonal fasciculation (Chiba, 1999 via Flybase). 22C10 is the monoclonal antibody for FUTSCH gene, which encodes futsch a microtubule association protein (MAP) expressed in subsets of axons in both the CNS and PNS, and involved in axonogenesis and negative regulation of the neuron apoptotic process (Bettencourt da Cruz et al., 2005).
Differential neuroanatomy mapped by detection of these integral neurodevelopmental proteins with known wild-type expression patterns should shed insight on the neurodevelopmental function & interaction of the vtd gene. Tracing these neurodevelopmental and neuroanatomical abnormalities in the Drosophila model will provide revealing clues about the homologous human gene attributable to a subset of non-classical Cornelia De Lange Syndrome.
METHOD
The experiment applies immunohistochemistry, anti-body staining with molecular markers, to visualize neural protein expression patterns in Drosophila neurodevelopment. Conducted on Drosophila melanogaster embryos in varying neurodevelopmental stages fixed in 4% formaldehyde, the investigation examined slides of 21 wild-type subjects and 23 vtd genetic mutants (created via balancer chromosome rescue) under immunofluorescent microscopy.
Monoclonal mouse antibodies selective for proteins FAS II in one analysis, and for 22c10 in a separate analysis, served as the primary antibodies applied to both the wild-type and the vtd mutant strain embryos.
Fluorescent dye horseradish peroxidase (HRP) of goat origin served as the secondary antibody to tag the primary mouse antibodies. The addition of N-propyl gallate subdues the degradation of the fluorescent signal from photobleaching. Experimental procedures were conducted in PBS-based buffer solution at pH 7.0, optimal to active antibodies.
Cell permeabilization prior to the addition of antibodies involved a triple-wash of embryos in PBS-Triton X (PBT) solution to rinse off the formaldehyde, rocking wash, and incubation on a rotator. To block non-specific binding, 4% goat serum was added with mouse anti-FAS II in one set of wild-type and mutant embryos, and mouse anti-22c10 in the second set, followed by rocking incubation overnight at 4˚C.
Next-day secondary antibody incubation with goat anti-mouse-HRP to identify the monoclonal mouse antibodies followed the repeated process of washing and rocking incubation at 4˚C. Immunofluorescent microscopy for enzymatic detection of FAS II and 22c10 expression in wild-type and mutant nervous systems utilized a diaminobenzidine (DAB) stain with 0.3% hydroxide solution.
A serial dehydration of the stained embryos with ethanol was conducted, followed by addition of methyl salicylate for storage. Embryos were mounted and oriented three or four at a time onto slides with methyl salicylate for viewing under compound microscope, then sealed with permount.
RESULTS & DISCUSSION
The fruits of the experimental immunohistochemistry resulted in a large number of wild-type and vtd mutant Drosophila embryos appropriate for separate FAS II and 22C10 analyses under light microscopy. Embryos with too much tissue wear or crushed during the mounting/orienting process, those with incomplete antibody binding/staining, and homozygous lethal mutants (for either balancer or vtd imprecise excision) displaying no protein expression, were excluded from the data. A sample of about 20 per subset, chosen from the mounted slides, were selected for comparative analysis—reflected in Table 1.
Registration of FAS II protein expression was tagged a brown color by DAB-staining with the secondary anti-mouse antibody. This visualization of the neuroanatomy of the ventral nerve cord in the Drosophila CNS can be observed in Appendix A, revealing the characteristic three axonal connectives running longitudinally parallel in wild-type Drosophila neurodevelopment. Experimentally, only two embryos, out of five from the stained stock observed under microscopy, were able to be successfully isolated and clearly displaying the classic FAS II expression pattern phenotype.
In contrast, vtd mutant embryos generally exhibited visibly less-pronounced FAS II axonal protein detection and diminished distinguishability of longitudinal axonal connectives (Appendices B-C). Whereas the majority of observed vtd mutants definitively lacked the wild-type phenotype in the ventral nerve cord (a là C), it remains unclear whether or not certain members exhibited two rather than three parallel connectives on the left side, or if these were undefined due to the appearance of constriction (Appendix B). Among the reservations in the experiment, were variation in developmental stages among subjects, unconfirmed/inferred genotype status in members of mutant sample, and possible variation in success of antibody-staining technique. We note these experimental limits because of the range in deviation from the known wild-type phenotype among vtd mutant embryos.
The inferred diminished expression of FASCILIN protein crucial to proper axonal fasciculation, visualized as leaky, weak axonal connectives, suggests a hypomorph mutation responsible for diminished axonal guidance in vtd mutants with defective cohesin. These outcomes are consistent with findings in RAD21 orthologs in C. elegans and zebrafish (Benard, 2004), elucidating a possible neurodevelopmental mechanism behind the severe cognitive disability in the CDLS variant characterized by RAD21 cohesinopathy. Our results further suggest a spectrum of abnormal cohesin activity levels and effective vtd mutation strength a là Cornelia de Lange phenotypes. Further investigation is required to confirm this can be traced to a genotypic basis, and not purely due to variation in staining penetrative efficacy. Regarding the spectrum of phenotypes, because vtd only encodes RAD21, one of three core cohesin components, neither knockout nor mutation should present null in cohesin expression, if SMC1A and SMC3—nevermind the cohesin regulatory interactions with Nipped-B/NIPBL— remain in play.
The second investigation with 22C10 antibody traced FUTSCH expression in the Drosophila embryonic central and peripheral nervous systems. FUTSCH wild-type expression distinguishes ventral unpaired median (VUM) neuron bodies protruding from the center of the ventral nerve cord, flanked by aCC motor neurons, from surrounding pockets of mesectodermal tissue in the CNS (Appendix D). VUM bodies and aCC motor neurons are also visible as a reference point in the futsch-selective CNS of vtd mutants (Appendices E-G). In vtd mutants, however, the CNS appears more congested with these axons and neurons than in their wild-type counterparts. Potential deviation in focal plane and variation in developmental stages among subjects should be noted.
A similar effect can be observed in the comparisons of peripheral nervous system neuroanatomy (visualized with 22C10 staining of futsch expression in Appendix J) in vtd mutants against known wild-type expression (Appendices K-L). Chordontal clusters appeared abnormally larger and greater in members per cluster, most notably visible in the irregular lateral clusters in the focal plane of Appendix J. These findings suggest vtd gene’s role in regulation of axonal apoptosis and pruning necessary for proper neurodevelopment, consistent with Drosophila investigations by Pauli 2008, which featured abolished developmental pruning of axons and dendrites in γ neurons due to RAD 21 cleavage. Not only do these observed neurodevelopmental abnormalities of defective RAD21 cohesinopathy shed insight the severe motor impairment and mental disability in CDLS, the experiment’s immunohistochemical investigation places FUTSCH as the MAP mechanism for this neurodevelopmental phenomenon of cohesinopathy, due to FUTSCH’s role as a negative regulator of the neuron apoptotic process (Bettencourt da Cruz et al., 2005).
