MHM plays a role in chicken embryonic development, including gonadogenesis

Definitions:

  • Avian: Birds, feathered, winged, bipedal, warm-blooded, egg-laying, vertebrate animals
  • ChickenGallus gallus domesticus- Domestic fowl and a subspecies of the Red Junglefowl
    • Habitat: Native to Southern Asia, particularily in the jungles of India
    • Life Span: Up to 30 years
    • Food Habits: Herbivore and Incectivore, can also eat corn, soybean, worms, grass and different types of grain.

OVERVIEW OF ARTICLE

The chicken Z chromosome-linked locus MHM, is an integral part of a chicken’s normal embryonic and gonadal development. It is methylated and transcriptionally silent in male cells, but is hypomethylated and transcribed into a long non-coding RNA in female cells. It has been linked to sex-determination in the chicken embryo but lacks evidence. Variations of the MHM gene in both males and females can cause a plethora of abnormalities in the gonads and other parts of the chicken embryo’s anatomy2.The purpose of this research is to investigate the role of this gene using different variants of mis-expression of the sense and antisense strands. Understanding the MHM gene can further elucidate characteristics of sexual determination in chickens, thus providing a better comparison with other model organisms.

BACKGROUND

Nicholson, Heather. Sex Determination in Chickens. July, 2012. WordPress.com. April 2014

Image 2. Nicholson, Heather. Sex Determination in Chickens. July, 2012. WordPress.com. April 2014

Sex determination of chicken embryos is determined by the ZZ (male) and ZW (female) chromosome system.The Z chromosome is linked with genes that determine embryonic and gonadal formation. The Z linked gene DMRT1 is the only known gene in sex determination in the avian system, especially in development of males3. The regions across the Z-chromosome are fully or partially compensated, so they harbor specific genes that are important for dosage inequality between sexes. MHM (Male Hypermethaylated) gene is a Z-linked gene that is hypermethylated and silent in male cells and expressed in female cells.

1999 Nature Publishing Group Nanda, I. et al. 300 million years of conserved synteny between chicken Z and human chromosome 9. Nature Genetics 21, 259 (1999).

Image 3. Visualization of DMTR1 in chicken embryo. 1999 Nature Publishing Group Nanda, I. et al. (a). Hybridization of DMRT1 cosmid to a female chicken metaphase spread. (b.) DAPI banding of the same metaphase spread, converted into G-like bands. (c). G-banded karyotype of chicken macrosomes hybridized with DMRT1 (green spot on Z chromosome).

MHM (Male HyperMethylated) gene

  • Permanently hypermethylated on the two Z sex chromosomes of male chickens and is transcriptionally silent.
  • It’s located on the single Z chromosome and is transcribed into a long non-coding RNA that coats the Z chromosome on site of transcription.
Nicholson, Heather. Chicken Chromosomes. July, 2012. WordPress.com. April 2014

Image 4. Nicholson, Heather. Chicken Chromosomes. July, 2012. WordPress.com. April 2014

EXPERIMENT PROCEDURES

In an effort to understand the role of this gene in gonadal development, the researchers carried out detailed expression and mis-expression studies. There were several experiments done on RNA transcription of both sense and mis-sense strands of the MHM locus, to further determine the temporal and spatial expression patterns within embryonic and gonadal development.

The experiment began by cloning a 2.2 kb MHM repeat unit using a polymerase chain reaction (PCR). The first analysis was a Northern Blot, this was done in order to study gene expression by detection of RNA (or isolated mRNA) in a sample. The total RNA used for the study was isolated from several embryonic male and female gonads & limb and brain samples. The next analysis was a whole mount in situ hybridization, which is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or section of tissue. For this portion of the experiment they studied the embryos, gonads, limbs, heart and brachial arch/brain. Next, they studied mis-expression of 2.2 kb MHM in ovo and further analyzed that data using RT-PCR. Finally, immunostaining and histological procedures were carried out in order to visually asses the effects of the MHM in embryonic, gonadal and brain tissue. All experiments were performed twice in order to ensure accuracy.

RESULTS

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Figure 1. Open reading frames showing MHM sense and anti-sense strands.

  • Overall this data showed female transcripts on both sense and antisense orientation
  • Male transcripts were not really found on either sequence
Figure 2. Northern blot analysis of embryonic gonad (G) limb (L) and Brain (B) in MHM sense strand expression (A) and MHM antisense strand expression (B)

Figure 2. Northern blot analysis of embryonic gonad (G) limb (L) and Brain (B) in MHM sense strand expression (A) and MHM antisense strand expression (B)

  •  Northern blot shows strong female-specific expression of MHM sense strand detected throughout the embryos.
  • MHM sense was expressed in the (female) gonads, with expression increasing over development
  • No MHM sense expression was detected in males
  • Antisense strand was also expressed but at lower levels with a more restricted pattern
Female-specific expression of MHMsense RNA, using either 0.668 kb or 2.2 kb riboprobe. (A) Whole embryo expression, delineated with a 2.2 kbMHM riboprobe. Expression in female embryos is detected first throughout from day 1. Strong expression is seen by day 4. Males do not show expression. (B)MHM expression in the urogenital system, using the 668 bp riboprobe. Expression is detectable in female gonads (G, outlined), in the M¨ ullerian duct (arrowhead) and at lower levels in the mesonephros (Ms), from day 4.5–8.5. Gonadal expression increases over time. No expression is detectable in male gonads

Figure 3. Female-specific expression of MHMsense RNA. (A) Whole embryo expression. (B)MHM expression in the urogenital system.

Female-specific expression ofMHM-antisenseRNA. (A)MHM-antisenseRNA expression in day 1 female embryos. At E4.5, expression is detectable in the telencephalon (Tel) and mesencephalon (Mes) of the brain, and in the foreand hindlimbs. Males did not show expression. (B) Low level-MHM-antisense expression in the gonads (G) outlined in white. Ms¼mesonephric kidneys. Weak expression observed from day 4.5 in the females with stronger expression observed at 6.5 and 8.5. Little expression is observed in the males. Scale bar¼50mm

Figure 4. Female-specific expression of MHM-antisense RNA.(A)MHM-antisense RNA expression in day 1 female embryos. (B) Low level-MHM-antisense expression in the gonads (G) outlined in white.

  • MHM sense RNA localization was detected throughout both left and right gonads and in mesonephric kidneys of female embryos.(Figure 3 and 4)
  • MHM sense strand was present in the cytoplasm of developing ovarian cortex of female gonads with weak staining detected in underlying medulla
    • Cortex: site of germ cell proliferation and folliculogenesis
    • Medulla: site of estrogen biosynthesis

MIS-EXPRESSION OF MHM IN MALE AND FEMALE EMBRYOS

Localization of MHM sense andantisensetranscripts in E10.5 gonads, using 0.668 kb or 2.2 kb riboprobe. (A) Punctate expression ofMHM-sensein a E6.5 female gonad and in the neighboring mesonephric kidney (insert shows nuclear localization). DAPI counter-staining confirms a nuclear localization in E10.5 ovary (arrows). (B)MHM-antisenseexpression in the cytoplasm of the left ovarian cortex. (C ) and in scattered medullary cells (M). The right gonad lacks a cortex and shows medullary staining only. False DAPI counterstaining for nuclei (blue) shows that MHM-Forward is cytoplasmic (red). Bar¼100 um. (C) No specific staining for either RNA in E10.5 testis.

Figure 5. Localization of MHM sense and antisense transcripts.(A) Punctate expression of MHM-sense in a female gonad and in the neighboring mesonephric kidney (insert shows nuclear localization). DAPI counter-staining confirms a nuclear localization in ovary(arrows). (B)MHM anti-sense expression in the cytoplasm of the left ovarian cortex. (C ) and in scattered medullary cells (M).

  • MHM isn’t normally expressed in male embryos, due to epigenetic silencing
  • MHM sense and antisense sequences, mis-expression perturbed testis development but did not induce feminisation
  • Higher levels of MHM antisense mis-expression caused a drop in DMRT1 levels compared to controls
  • MHM also increased male-biased embryo mortality
Lack of gonadal asymmetry and production of bilateral ovaries in MHM sense and antisense infected female embryos. Aromatase enzyme immunofluorescence delineated bilateral enlarged ovaries. Viral P27 staining confirmed robust virus infection in these gonads. UGS¼urogenital system; lg¼left gonad; Rg¼right goand; Ms¼mesonephric kidneys

Figure 6. Effects of MHM-sense and antisense mis-expression in female chicken embryos. Lack of gonadal asymmetry and production of bilateral ovaries in MHM sense and antisense infected female embryos.

  •  Mis-expression of MHM-sense induced abnormalities the size and placement of the female gonads
  • This resulted in the development of bilateral enlarged ovaries
  • Significantly larger than normal asymmetric female gonads
  • However, this did not increase female embryo mortality

CONCLUSION

  • Overall MHM plays a potential role in regulating embryonic growth and gonadal development
  • In females, mis-expression of the gene can cause phenotypic changes in the development of the ovaries and other gonadal regions
  • In males, mis-expression of the gene doesn’t cause direct harm to gonadal development, but it does cause a significant drop in DMRT1 mRNA levels which in turn cause developmental problems and can even cause death in some cases4.

DISCUSSION

The MHM gene plays a vital role in development in many aspects of the chicken’s anatomy. In this experiment normal expression of sense and antisense strands were transcribed and analyzed against mis-expression. However, in order to fully understand the impact this has on chicken development, more genetic variation experiments will have to take place, such as increasing genetic expression or deletion of the gene entirely5. These tests will further confirm the MHM and its effect on female and male gonadal development.

CRITIQUE

Overall, the paper was very interesting, the strength of the study stemmed from all the various methods of analysis. I did not only tie the research together, but it adequately contributed support of each of the observations made regarding the MHM gene. The different figures helped to personify how the mis -expression affected not only the embryo, but also other parts of the chicken’s anatomy. However, I felt that there could have been more genetic analysis is on the specific location and the dosage of the gene. The reason for this, is that not only can the appearance of the gene cause a change in the gonadal and embryonic development of the chicken; the amount/dosage level of the gene represented on the chromosome can also add to the expression levels within the developing organism. This analysis could have definitely been incorporated better into the research, in order to further round out the gonadal and embryonic analysis.

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Image 5. Dancing Chicken. ClipArtBest.com April 2014

THAT’S ALL FOLKS

References 

  1.  “Gallus Gallus — Details.” Encyclopedia of Life. N.p., n.d. Web. 03 Apr. 2014.
  2. Nicholson, Heather. “GMS5: Chicken Chromosomes.” Scratch Cradle. N.p., July 2012. Web. Apr. 2014.
  3.  Hake, L. & O’Connor, C. (2008) Genetic mechanisms of sex determination. Nature Education 1(1):25
  4.  Roeszler, Kelly N., Catherine Itman, Andrew H. Sinclair, and Craig A. Smith. “The Long Non-coding RNA, MHM, Plays a Role in Chicken Embryonic Development, including Gonadogenesis.” Developmental Biology 366.2 (2012): 317-26. Print.
  5. Teranishi, M., Shimada, Y., Hori, T., Nakabayashi, O., Kikuchi, T., Macleod, T., Pym, R., Sheldon, B., Solovei, I., Macgregor, H., Mizuno, S., 2001. Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjcent to theDMRT1 locus. Chromosome Res. 9, 147–165    

 

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