Keng Yih Chew

Antibodies Mediate Formation of Neutrophil Extracellular Traps in the Middle Ear and Facilitate Secondary Pneumococcal Otitis Media

ABSTRACT Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

HOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii

BackgroundKangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby.ResultsWe describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken.ConclusionsThis is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.

Heterochrony in the regulation of the developing marsupial limb

Background: At birth, marsupial neonates have precociously developed forelimbs. The development of the tammar wallaby (Macropus eugenii) hindlimbs lags significantly behind that of the forelimbs. This differs from the grey short‐tailed opossum, Monodelphis domestica, which has relatively similar fore‐ and hindlimbs at birth. This study examines the expression of the key patterning genes TBX4, TBX5, PITX1, FGF8, and SHH in developing limb buds in the tammar wallaby. Results: All genes examined were highly conserved with orthologues from opossum and mouse. TBX4 expression appeared earlier in development than in the mouse, but later than in the opossum. SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. Conclusions: The most marked divergence in limb size in marsupials occurs in the kangaroos and wallabies. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs. Developmental Dynamics 243:324–338, 2014.

Hormone-Independent Pathways of Sexual Differentiation

New observations over the last 25 years of hormone-independent sexual dimorphisms have gradually and unequivocally overturned the dogma, arising from Josts elegant experiments in the mid-1900s, that all somatic sex dimorphisms in vertebrates arise from the action of gonadal hormones. Although we know that Sry, a Y-linked gene, is the primary gonadal sex determinant in mammals, more recent analysis in marsupials, mice, and finches has highlighted numerous sexual dimorphisms that are evident well before the differentiation of the testis and which cannot be explained by a sexually dimorphic hormonal environment. In marsupials, scrotal bulges and mammary primordia are visible before the testis has differentiated due to the expression of a gene(s) on the X chromosome. ZZ and ZW gynandromorph finches have brains that develop in a sexually dimorphic way dependent on their sex chromosome content. In genetically manipulated mice, it is the X chromosomes, not the gonads, that determine many characters including rate of early development, adiposity, and neural circuits. Even spotted hyenas have sexual dimorphisms that cannot be simply explained by hormonal exposure. This review discusses the recent findings that confirm that there are hormone-independent sexual dimorphisms well before the gonads begin to produce their hormones.

A Dual Role for SHH during Phallus Development in a Marsupial

The mammalian phallus arises from identical primordia in both sexes and is patterned in part by the key morphogen Sonic hedgehog (SHH). We have investigated SHH and other morphogens during phallus development in the tammar wallaby. In this marsupial, testis differentiation and androgen production occurs just after birth, but it takes a further 50-60 days before the phallus becomes sexually dimorphic. One day before birth, SHH was expressed in both sexes in the urethral epithelium. In males, there was a marked upregulation of SHH, GLI2, and AR at day 50 postpartum, a time when testicular androgen production falls. SHH, GLI2, and AR were downregulated in female pouch young treated with androstanediol from days 24-50, but not when treatments were begun at day 29, suggesting an early window of androgen sensitivity. SHH, GLI2, and AR expression in the phallus of males castrated at day 23 did not differ from controls, but there was an increase in SHH and GLI2 and a decrease in FGF8 and BMP4 expression when the animals were castrated at day 29. These results suggest that the early patterning by SHH is androgen-independent followed by an androgen-dependent window of sensitivity and a sharp rise in SHH expression after androgen withdrawal at day 50.

FOXA1 and SOX9 Expression in the Developing Urogenital Sinus of the Tammar Wallaby (Macropus eugenii)

The mammalian prostate is a compact structure in humans but multi-lobed in mice. In humans and mice, FOXA1 and SOX9 play pivotal roles in prostate morphogenesis, but few other species have been examined. We examined FOXA1 and SOX9 in the marsupial tammar wallaby, Macropus eugenii, which has a segmented prostate more similar to human than to mouse. In males, prostatic budding in the urogenital epithelium (UGE) was initiated by day 24 postpartum (pp), but in the female the UGE remained smooth and had begun forming the marsupial vaginal structures. FOXA1 was upregulated in the male urogenital sinus (UGS) by day 51 pp, whilst in the female UGS FOXA1 remained basal. FOXA1 was localised in the UGE in both sexes between day 20 and 80 pp. SOX9 was upregulated in the male UGS at day 21-30 pp and remained high until day 51-60 pp. SOX9 protein was localised in the distal tips of prostatic buds which were highly proliferative. The persistent upregulation of the transcription factors SOX9 and FOXA1 after the initial peak and fall of androgen levels suggest that in the tammar, as in other mammals, these factors are required to sustain prostate differentiation, development and proliferation as androgen levels return to basal levels.

Inducing sex reversal of the urogenital system of marsupials

Marsupials differ from eutherian mammals in their reproductive strategy of delivering a highly altricial young after a short gestation. The young, with its undeveloped organ systems completes its development post-natally, usually within a pouch. The young is dependent on milk with a composition that varies through lactation to support its growth and changing needs as it matures over a lengthy period. Gonadal differentiation occurs after birth, providing a unique opportunity to examine the effects of hormonal manipulations on its sexual differentiation of the highly accessible young. In marsupials a difference in the migration of the urinary ducts around the genital ducts from eutherian mammals results in the unique tammar reproductive tract which has three vaginae and two cervices, and two distinctly separate uteri. In the tammar wallaby, a small member of the kangaroo family, we showed that virilisation of the Wolffian duct, prostate and phallus depends on an alternate androgen pathway, which has now been shown to be important for virilisation in humans. Through hormonal manipulations over differing time periods we have achieved sex reversal of both ovaries and testes, germ cells, genital ducts, prostate and phallus. Whilst we understand many of the mechanisms behind sexual differentiation there are still many lessons to be learned from understanding how sex reversal is achieved by using a model such as the tammar wallaby. This will help guide investigations into the major questions of how and why sex determination is achieved in other species. This review discusses the control and development of the marsupial urogenital system, largely drawn from our studies in the tammar wallaby and our ability to manipulate this system to induce sex reversal.

Expression of STRA8 is conserved in therian mammals but expression of CYP26B1 differs between marsupials and mice.

Abstract The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown. Summary Sentence This comparative study using a marsupial species suggests that the roles of retinoic acid and STRA8 but not CYP26B1 in controlling the initiation of meiosis are conserved between different mammals.