Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Inversin (NPHP2) and Vangl2 are required for normal zebrafish cloaca formation.

Nephronophthisis (NPH), an autosomal recessive ciliopathy, results from mutations in more than 20 different genes (NPHPs). These gene products form protein complexes that regulate trafficking within the cilium, a microtubular structure that plays a crucial role in developmental processes. Several NPHPs, including NPHP2/Inversin, have been linked to extraciliary functions. In addition to defining a specific segment of primary cilia (Inversin compartment), NPHP2 participates in planar cell polarity (PCP) signaling along with Dishevelled and Vangl family members. We used the mutant zebrafish line invssa36157, containing a stop codon at amino acid 314, to characterize tissue-specific functions of zebrafish Nphp2. The invssa36157 line exhibits mild ciliopathy phenotypes and increased glomerular and cloaca cyst formation. These mutants showed enhanced susceptibility to the simultaneous depletion of the nphp1/nphp2/nphp8 module, known to be involved in the cytoskeletal organization of epithelial cells. Notably, simultaneous depletion of zebrafish nphp1 and vangl2 led to a pronounced increase in cloaca malformations in the invssa36157 mutant embryos. Time-lapse imaging showed that the pronephric cells correctly migrated towards the ectodermal cells in these embryos, but failed to form the cloaca opening. Despite these abnormal developments, cellular fate does not seem to be affected in nphp1 and vangl2 MO-depleted invssa36157 mutants, as shown by in situ hybridizations for markers of pronephros and ectodermal cell development. However, significantly reduced apoptotic activity was observed in this double knockdown model, signifying the role of apoptosis in cloacal morphogenesis. Our findings underscore the critical interplay of nphp1, nphp2/Inversin, and vangl2 in orchestrating normal cloaca formation in zebrafish, shedding light on the complex molecular mechanisms underlying ciliopathy-associated phenotypes.

A teleost structural analogue to the avian bursa of Fabricius.

The bursa of Fabricius is a primary and secondary lymphoid organ considered exclusively present in birds, and studies of this structure have been vital to our current understanding of the adaptive immune system of vertebrates. In this study, we reveal substantial lymphoepithelial tissue in a previously undescribed bursa in Atlantic salmon (Salmo salar), situated caudal to the urogenital papilla of the cloaca and thus analogous to the anatomical placement of the bursa of Fabricius. We investigated three groups of Atlantic salmon at different maturational stages and characterized the structure by applying dissection, radiology, scanning electron microscopy and histological techniques, including immunohistochemistry and in situ hybridization. We found that the epithelial anlage of the salmon cloacal bursa developed into substantial lymphoepithelial tissue and subsequently regressed following sexual maturation. Such a dynamic development is also a key characteristic of the avian bursa. The presence of intraepithelial lymphocytes was concomitant with expression of the leukocyte-attracting chemokine CCL19, indicative of lymphoid organ functions. We did not observe recombination or gene conversion in salmon bursal lymphocytes at any developmental stage, indicating the absence of primary lymphoid organ functions in contrast to the bursa of Fabricius. However, the possibility of the bursa to trap both enteric and environmental antigens, combined with the presence of several antigen-presenting cells residing within the lymphoepithelium, suggest the structure has secondary lymphoid organ functions. We present the discovery of a lymphoid organ in Atlantic salmon with striking topographical similarities to that of the bursa of Fabricius in birds. In addition, the age-dependent dynamics of its lymphoepithelium suggest functions related to the maturation processes of lymphocytes.

Salt-water acclimation of the estuarine crocodile Crocodylus porosus involves enhanced ion transport properties of the urodaeum and rectum.

Estuarine crocodiles, Crocodylus porosus, inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na+/K+-ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na+ channel (ENaC) and/or the apical Na+/H+ exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18 ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments.

Reciprocal Spatiotemporally Controlled Apoptosis Regulates Wolffian Duct Cloaca Fusion.

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLCγ docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion in vivo Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT.

The Effects of Capture, Restraint, and Transport on Hematologic, Plasma Biochemical, and Blood Gas Values in Dalmatian Pelicans ( Pelecanus crispus).

To determine the effect of capture and restraint on hematologic, plasma biochemical, and venous blood gas values in Dalmatian pelicans ( Pelecanus crispus), blood samples were collected and temperature and heart rate were recorded in 13 pelicans (5 males and 8 females) immediately after capture from a large field exhibit. Repeat sampling was performed after transport, a mean of 74 minutes after initial evaluation. When compared with values at initial sampling to the time of capture, significant increases were documented in the heterophil : lymphocyte ratio (H : L), carbon dioxide (CO2), bicarbonate (HCO3), total carbon dioxide (Tco2), base excess (BE), and partial pressure of carbon dioxide (Pco2) values after 74 minutes. Significant decreases were documented in total protein, lactate, and phosphorus concentrations, percentage of oxygen saturation (So2), partial pressure of oxygen (Po2), total white blood cell count (WBC), absolute lymphocytes, absolute eosinophils, and absolute monocytes after 74 minutes. Those changes suggest capture and restraint can alter hematologic, plasma biochemical, and acid-base status in Dalmatian pelicans, which may influence clinical decision making and case management.

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals.

Selective constraint dominates the evolution of genes expressed in a novel reproductive gland.

One striking pattern in molecular evolution is that genes encoding proteins involved in reproduction tend to evolve rapidly. Seminal fluid proteins frequently exhibit this pattern and directly affect multiple reproductive processes including enhancing sperm performance and mediating postmating sexual selection. Here, we investigate molecular evolutionary patterns of genes expressed in the foam gland of Japanese quail (Coturnix japonica), a novel reproductive phenotype. Foam provides an interesting contrast to seminal fluid because it plays a similar functional role, yet is produced, stored, and transferred to females independent of semen. We combined RNA-Seq and comparative genomics to examine evolutionary rates of genes with enriched expression in the foam gland of Japanese quail and those that exhibit enriched expression in two other tissues (testis and liver) and with broadly expressed genes. Overall, we found pronounced heterogeneity in evolutionary rates. Foam gland genes evolved under strong evolutionary constraint, whereas testis genes evolved rapidly and sometimes adaptively. These striking differences were robust to variation in gene expression. Genes with enriched expression in the foam gland did not show major shifts in selective pressure after the quail and chicken lineages split; in contrast, testis-expressed genes experienced a burst of accelerated evolution specifically along the Coturnix lineage. Our work demonstrates that, as a class, genes expressed in the novel foam gland experience different selection regimes than genes expressed in many other tissues producing seminal fluid proteins. Our results also highlight the importance of selective constraint in shaping the evolution of male reproductive genes.

Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice.

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.

Proteomic analysis reveals dynamic changes in cloacal fluid composition during the reproductive season in a sexually promiscuous passerine.

Cryptic female choice (CFC) is a component of postcopulatory sexual selection that allows females to influence the fertilization success of sperm from different males. While its precise mechanisms remain unclear, they may involve the influence of the protein composition of the female reproductive fluids on sperm functionality. This study maps the protein composition of the cloacal fluid across different phases of female reproductive cycle in a sexually promiscuous passerine, the barn swallow. Similar to mammals, the protein composition in the female reproductive tract differed between receptive (when females copulate) and nonreceptive phases. With the change in the protein background, the enriched gene ontology terms also shifted. Within the receptive phase, distinctions were observed between proteomes sampled just before and during egg laying. However, three proteins exhibited increased abundance during the entire receptive phase compared to nonreceptive phases. These proteins are candidates in cryptic female choice, as all of them can influence the functionality of sperm or sperm-egg interaction. Our study demonstrates dynamic changes in the cloacal environment throughout the avian breeding cycle, emphasizing the importance of considering these fluctuations in studies of cryptic female choice.

Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.

NPHP4 mutations cause nephronophthisis, an autosomal recessive cystic kidney disease associated with renal fibrosis and kidney failure. The NPHP4 gene product nephrocystin-4 interacts with other nephrocystins, cytoskeletal and ciliary proteins; however, the molecular and cellular functions of nephrocystin-4 have remained elusive. Here we demonstrate that nephrocystin-4 is required for normal cloaca formation during zebrafish embryogenesis. Time-lapse imaging of the developing zebrafish pronephros revealed that tubular epithelial cells at the distal pronephros actively migrate between the yolk sac extension and the blood island towards the ventral fin fold to join the proctodeum and to form the cloaca. Nphp4-deficient pronephric duct cells failed to connect with their ectodermal counterparts, and instead formed a vesicle at the obstructed end of the pronephric duct. Nephrocystin-4 interacts with nephrocystin-1 and Par6. Depletion of zebrafish NPHP1 (nphp1) increased the incidence of cyst formation and randomization of the normal body axis, but did not augment cloaca malformation in nphp4-deficient zebrafish embryos. However, simultaneous depletion of zebrafish Par6 (pard6) aggravated cloaca formation defects in nphp4-depleted embryos, suggesting that nphp4 orchestrates directed cell migration and cloaca formation through interaction with the Par protein complex.

Six1 and Eya1 are critical regulators of peri-cloacal mesenchymal progenitors during genitourinary tract development.

The evolutionarily conserved Six1-Eya1 transcription complex is central to mammalian organogenesis, and deletion of these genes in mice results in developmental anomalies of multiple organs that recapitulate human branchio-oto-renal (BOR) and DiGeorge syndromes. Here, we report that both Six1 and Eya1 are strongly expressed in the peri-cloacal mesenchyme (PCM) surrounding the cloaca, the terminal end of hindgut dilation. Six1 and Eya1 are absent from the intra-cloacal mesenchyme (ICM), a cell mass that divides the cloaca into dorsal hindgut and ventral urogenital sinus. Deletion of either or both Six1 and Eya1 genes results in a spectrum of genitourinary tract defects including persistent cloaca - hypoplastic perineum tissue between external urogenital and anorectal tracts; hypospadias - ectopic ventral positioning of the urethral orifice; and hypoplastic genitalia. Analyses of critical signaling molecules indicate normal expression of Shh in the cloaca and cloaca-derived endodermal epithelia. Using a Cre/loxP genetic fate mapping strategy, we demonstrate that Six1-positive PCM progenitors give rise to the most caudal structures of the body plan including the urogenital and anorectal complex, and the perineum region. Thus, Six1 and Eya1 are key regulators of both upper and lower urinary tract morphogenesis. Results from this study uncover essential roles of the PCM progenitors during genitourinary tract formation.

Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development.

Malformations of the external genitalia are among the most common congenital anomalies in humans. The urogenital and anorectal sinuses develop from the embryonic cloaca, and the penis and clitoris develop from the genital tubercle. Within the genital tubercle, the endodermally derived urethral epithelium functions as an organizer and expresses sonic hedgehog (Shh). Shh knockout mice lack external genitalia and have a persistent cloaca. This identified an early requirement for Shh, but precluded analysis of its later role in the genital tubercle. We conducted temporally controlled deletions of Shh and report that Shh is required continuously through the onset of sexual differentiation. Shh function is divisible into two temporal phases; an anogenital phase, during which Shh regulates outgrowth and patterning of the genital tubercle and septation of the cloaca, and a later external genital phase, during which Shh regulates urethral tube closure. Disruption of Shh function during the anogenital phase causes coordinated anorectal and genitourinary malformations, whereas inactivation during the external genital phase causes hypospadias. Shh directs cloacal septation by promoting cell proliferation in adjacent urorectal septum mesenchyme. Additionally, conditional inactivation of smoothened in the genital ectoderm and cloacal/urethral endoderm shows that the ectoderm is a direct target of Shh and is required for urethral tube closure, highlighting a novel role for genital ectoderm in urethragenesis. Identification of the stages during which disruption of Shh results in either isolated or coordinated malformations of anorectal and external genital organs provides a new tool for investigating the etiology of anogenital malformations in humans.

Scent chemicals of the brushtail possum, Trichosurus vulpecula.

The common brushtail possum (Trichosurus vulpecula) is the most widespread browsing marsupial in Australia, where it occupies woodland, agricultural, and urban environments. Following its introduction into New Zealand in the 19th century it has become a major feral pest, threatening native forests. The adaptability of the possum is thought to be due in part to its social organization, in which chemical communication is important. Possums have cloacal glands and exhibit related marking behavior. This study sought to characterize the chemicals involved in scent marking. Swabs were taken of the cloacal region of 15 possums (5 females, 10 males) from north-eastern Tasmania and analyzed by gas chromatography-mass spectrometry. There was a large number of compounds present, including 81 branched and unbranched, and saturated and unsaturated, fatty acids (C(4)-C(15)) and alcohols (C(6)-C(26)); 27 esters of 2,6- and 2,7-dimethyloctanol; 29 esters of formic acid; 39 sulfur compounds including S(8) and a series of dialkyl disulfides, trisulfides, and tetrasulfides (C(4)-C(10)); and several alkylglycerol ethers. Many of these cloacal compounds are new to biology. There was considerable individual variability in the relative amounts of compounds found, and no evident sex differences, although the study was not designed to test this. This pattern suggests that these compounds may be acting collectively as a signature mixture of semiochemicals, carrying information on the individual, its kinship, and physiological and social status. This is the first detailed description of putative semiochemicals in any marsupial species.

Spatiotemporal expression of Wnt5a during the development of the hindgut and anorectum in human embryos.

PURPOSE: The aim of the study was to determine the spatiotemporal expression of Wnt5a during hindgut and anorectum development in human embryos and to explore the possible role of Wnt5a during the morphogenesis of the human hindgut and anorectum. MATERIALS AND METHODS: The embryos (n = 107) were sectioned serially and sagittally, using Wnt5a immunohistochemical staining on the caudal midline from the 4th-9th weeks of gestation. RESULTS: From the 4th-7th week of gestation, the Wnt5a-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum and the urogenital sinus (UGS) opened to the amniotic cavity during the 7th week, the Wnt5a-positive cells disappeared and remained negative up to the 9th week on the epithelium of the anal canal. CONCLUSIONS: The expression of Wnt5a was constantly active during human hindgut and anorectum development and disappeared after the anus formed, suggesting that Wnt5a plays an important role in human hindgut and anorectal morphogenesis.

A novel role of CXCR4 and SDF-1 during migration of cloacal muscle precursors.

The cloaca acts as a common chamber into which gastrointestinal and urogenital tracts converge in lower vertebrates. The distal end of the cloaca is guarded by a ring of cloacal muscles or sphincters, the equivalent of perineal muscles in mammals. It has recently been shown that the development of the cloacal musculature depends on hindlimb muscle formation. The signaling molecules responsible for the outward migration of hindlimb myogenic precursors are not known. Based on the expression studies for CXCR4 and SDF-1, we hypothesized a role of this signaling pair during cloacal muscle precursor migration. The aim of our study was to investigate the role of SDF-1/CXCR4 during cloacal muscle precursor migration in the chicken embryos. We show that SDF-1 is expressed in the cloacal region, and by experimentally manipulating the SDF-1/CXCR4 signaling, we can show that SDF-1 guides the migration of CXCR4-expressing cloacal muscle precursors.

Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds.

Clade 2.3.4.4, H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused devastating effects across wild and domestic bird populations. We investigated differences in the intensity and distribution of the hemagglutinin (HA) glycoprotein binding of a clade 2.3.4.4 H5 HPAIV compared to a H5 low pathogenic avian influenza virus (LPAIV). Recombinant HA from gene sequences from a HPAIV, A/Northern pintail/Washington/40964/2014(H5N2) and a LPAIV, A/mallard/MN/410/2000(H5N2) were generated and, via protein histochemistry, HA binding in respiratory, intestinal and cloacal bursal tissue was quantified as median area of binding (MAB). Poultry species, shorebirds, ducks and terrestrial birds were used. Differences in MAB were observed between the HPAIV and LPAIV H5 HAs. We demonstrate that clade 2.3.4.4 HPAIV H5 HA has a broader host cell binding across a variety of bird species compared to the LPAIV H5 HA. These findings support published results from experimental trials, and outcomes of natural disease outbreaks with these viruses.

Bmp7 expression and null phenotype in the urogenital system suggest a role in re-organization of the urethral epithelium.

Signaling by Bone morphogenetic proteins (Bmps) has multiple and diverse roles in patterning and morphogenesis of the kidney, eye, limbs and the neural tube. Here, we employed the Bmp7(lacZ) strain to perform a detailed analysis of Bmp7 expression and the null phenotype during development of the mouse urogenital system. The urethral compartment originates in mid-embryogenesis from the ventral part of the cloaca, a transient cavity at the caudal end of the hindgut. At mid-gestation, Bmp7 expression was detected within several specific domains in the cloacal epithelium and mesenchyme. In late embryogenesis, Bmp7 expression was present in the urethra, rectum, the urethral glands, corpus cavernosum, and in the male and female genital ducts. Importantly, loss of Bmp7 resulted in arrest in cloacal septation, and severe defects in morphogenesis of the genital urethra and mesenchyme. Together, our analysis of Bmp7 expression and the null phenotype, indicates that Bmp7 may play an important role in re-organization of the epithelium during cloacal septation and morphogenesis of the genital tubercle.