Background: The purpose of this study is to characterize a full-term conjoined twins’ cadaver curated by Dr. Jacob Henle sometime between 1844 and 1852 and demonstrate digital distribution of an old and rare medical museum specimen using an extended reality (XR) model workflow. Methods: The cadaver (Preparation 296) is in the Department of Anatomy and Cell Biology at the University of Heidelberg. An XR display workflow comprises image capture, segmentation, and visualization using CT/MR scans derived from the cadaver. Online radiology presentation to medical students focuses on diagnostic characteristics of anatomical systems depicted with XR models. Results: Developmental defects in Preparation 296 include duplicated supradiaphragmatic structures and abnormal osteological features. Subdiaphragmatically, the gut is continuous on the right, but terminates at the distal esophagus on the left. One large liver occupies the abdomen with one spleen located on the left side. Observations suggest duplication of the primitive streak and separate notochords rostrally. Duplication occurs near the yolk sac and involves midgut formation while secondary midline fusion of the upper extremities and ribs likely results from the proximity of the embryos during development. Medical students access the model with device agnostic software during the curricular topic “Human Body Plan” that includes embryology concepts covering mechanisms of twinning. Conclusions: The workflow enables ease-of-access XR visualizations of an old and rare museum specimen. This study also demonstrates digital distribution and utilization of XR models applicable to embryology education.

Anatomical analysis of conjoined twins provides important information concerning embryological development. Museum specimens provide a unique sample that can be analyzed non-intrusively using advanced biomedical imaging and displayed online which is currently important due to limited in-person learning opportunities as a result of the COVID-19 pandemic. The purpose of this study is to create an extended reality (XR) workflow for visualization of dicephalic parapagus full-term conjoined twins obtained by Dr. Jacob Henle sometime between 1844-1852 for use in anatomy education. The workflow comprised image capture, segmentation, and visualization. The cadaver twins were curated at the University of Heidelberg and were subjected to CT and MR imaging. Relevant bones and soft tissues were manually segmented to create XR models, post-processed for visualization using Unity-based systems. A learning module was created and posted to Rad3d.com for presentation to students including visualization on Z-space computers (zspace.com) and sketchfab.com online. Osteology analysis showed commonality in the upper limb and shared ribs. Two vertebral columns were identified, and a single pelvic girdle was present with a single set of lower limbs. Duplicated supradiaphragmatic structures included two hearts (one with situs inversus) and four lungs, but single subdiaphragmatic visceral organs were observed. In particular, the gut was continuous on the right, but terminated at the distal esophagus on the left. One large liver occupied the abdomen with one large spleen located on the left. These observations suggest zygote fission was blocked near the yolk sac during midgut formation, but with secondary fusion of midline upper extremities and ribs. A radiology report was developed and presented to medical students as an embryology clinical correlation. This application indicates that university based museum specimens are useful as supplemental instructional subjects and, in this case, almost 170 years after arriving in the Department of Anatomy at the University of Heidelberg.

Objectives: The CL/Fr mouse displays cleft lip and palate (CLP) at a rate of 35%. The clf1 mutation is associated with CLP in related “A” strain mice and affects the gene Wnt9b. The purpose of this study was to determine tissue specific expression of Wnt9b during facial prominence morphogenesis in CL/Fr mice and provide new details concerning gene variants associated with CLP. Methods: Facial prominences from CLP(-) and CLP(+) CL/Fr and 3H1 wild-type (WT) mice at embryonic day 11.5 (E11.5) were collected for expression assays (DNA microarray analysis, qRT-PCR, immunostaining, and in situ hybridization). A modified Chi square test was used to analyze microarray data while a student t-test was used to statistically compare qRT-PCR values (p<0.05). Results: There was a partial and variable loss of Wnt9b in facial prominences of E11.5 CLP susceptible CL/Fr mice, with a greater loss associated with CLP(+). Two genes in the clf2 locus, Adcy2 and Ube2q11 also showed decreased expression. Two regulators of palatogenesis, Runx2 and Osr2 were significantly downregulated, while an inhibitor of cell proliferation, somatostatin (Sst), was elevated in CLP(+) relative to CLP(-) mice. Conclusion: Results indicate a role for Wnt9b in the pathogenesis of CLP and supports previous reports concerning its involvement with CLP in “A” strain mice. Misexpression of Sst suggests that it may be a downstream target of Wnt9b causing reduced overall growth possibly hindering fusion of facial prominences and contributing to the development of CLP.

BACKGROUND Cephalothoracopagus twinning is extremely rare, and it is characterized by fusion of the head and thorax, two separate spines, pelves, and fore- and hindlimbs. CASE In this case study, we describe cephalothoracopagus twinning in an embryonic mouse displaying a large but exencephalic head, median facial cleft, a single eye, and a second hindbrain rotated roughly 90° from a second spinal cord. There is a bony connection joining the clavicles, resulting in merged asternal thoracic cavities containing two hearts and four lungs. The abdominal cavities contain double caudal digestive tract structures, but a single esophagus and stomach. CONCLUSION There are several proposed theories regarding the mechanism of spontaneous conjoined twinning; however, the specific mechanisms are still largely unknown. In this report, we highlight the morphological features in a murine example of cephalothoracopagus twinning, furthering our understanding of this rare occurrence while also demonstrating developmental morphogenesis consistent with that reported for human conjoined twins.

The Brachyrrhine (Br) mutant mouse displays heritable frontonasal dysplasia (FND) and renal hypoplasia (RH). Previous DNA microarray analysis on embryonic Br facial explants showed a misexpression of several genes, two of which are Esrp1 and Esrp2. Together, the Esrp genes play an essential role in the regulation of epithelial‐mesenchymal transition—an important process in craniofacial and kidney development. The primary objective of this study is to determine if Esrp1 and ‐2 contributes to a developmental pathway leading to FND or RH in the Br mouse and, specifically, if there is a difference in Esrp1 and ‐2 expression in the developing midface and kidneys of wild type (WT) and Br adults and embryos. WT and Br/Br E11.5 medial nasal prominences (MNP) and E15.5 and E18.5 kidneys were dissected and RNA extracted for qPCR expression analysis. Preliminary results show minimal difference in Esrp1 and ‐2 expression in MNPs of WT and Br embryos, and an increase in Esrp2 expression from E15.5 to E18.5 WT kidneys. Future work involving qPCR and immunohistochemistry will be completed to better understand the expression patterns of these two genes. Results from this study will contribute to our understanding of the genetic basis of craniofacial and kidney morphogenesis.Grant Funding Source: 1R01‐DK‐064752 (SL)

The CL/Fr mouse demonstrates heritable bilateral and unilateral cleft lip and palate (CLP) at a rate of approximately 35%, generally above the background “A” strain mouse. Using classical mouse breeding strategies, it has been suggested that at least two disease loci, clf1 and clf2, are involved in the defect and candidate genes have been identified. Additionally, gene‐targeting analyses strongly suggest that Wnt9b contributes to CLP in the “A” strain mice. The aim of this study was to test the expression of clf1 and clf2 candidate genes in the facial prominences of CL/Fr embryos, utilizing microarray analysis. Medial nasal, lateral nasal, and maxillary prominences of phenotypically normal as well as cleft E11.5 CL/Fr mice were dissected and RNA was extracted using standard techniques for Agilent‐microarray protocol. Results indicate that expression of the clf1 candidate gene, Wnt9b, and the clf2 candidate gene, Ube2ql1, are significantly reduced (−3.11 and −1.83 fold, respectively) in the CL/Fr cleft tissues, suggesting these genes may be involved in the CLP mutation in CL/Fr mice. Future gene expression studies through quantitative RT‐PCR and regional expression analyses through immunohistochemistry and whole mount in situ hybridization will be performed to further test the expression of these clf1 and clf2 candidate genes.Grant Funding Source: NIH/NCRR 5P20RR024206 (S.J.S) and R01‐DK‐064752 (SL)

We have previously described the Br mutant mouse displaying heritable frontonasal dysplasia. Linkage analysis mapped the mutation near the homeobox transcription factor six2, normally expressed in the facial mesenchyme during development. The purpose of this study is to determine expression patterns of six2, as well as possible upstream and downstream targets of six2, in the developing midface. The three sets of facial prominences (medial, lateral, and maxillary) of E11.5 embryos were dissected and RNA extracted for qPCR assays and microarray analysis. Medial nasal prominences (MNP) were also taken for cell culture. qPCR results indicated six2 expression is highest in the MNP and demonstrated haploinsufficient down‐regulation in each of the three facial prominence sets in the Br mouse. Microarray results suggested the misregulation of several genes in the Br midface, including another member of the Six family of transcription factors: six3. qPCR and immunohistochemistry for six3 substantiated its upregulation in the microarray. Future work using RNA interference on six2 will be used to investigate possible pathways involving six2 and six3. Preliminary results using an in vitro knockdown of six2, performed on an MNP cell culture system utilizing siRNA, demonstrated a 65% knockdown of six2. These results may enable further in vitro work in order to elucidate a pathway in the developing midface involving six2. Grant Funding Source : NIH R01DK064752 (SL) & NCRR 5P20RR024206 (SJS)

Suboptimal kidney development resulting from a genetic deficit in nephron number can have lifelong consequences that may lead to cardiorenal complications upon exposure to secondary insults in later life. To determine whether the inherited reduced renal reserve compromises the ability to handle osmotic stress in the adult animal, we challenged the heterozygous 3H1 Brachyrrhine (Br/+) mouse, which displays heritable renal hypoplasia associated with reduced embryonic six2 expression, to a solution of 2% NaCl for 5 days or to fluid restriction for 48 h. Blood chemistry, fluid intake, and physiological parameters, including renal measurements, were determined. Systemic hypertonicity by prolonged salt loading led to significant increases in plasma osmolality and plasma Na(+), along with polydipsia and polyuria, with a significant urine-concentrating defect that was resistant to DDAVP treatment in the adult Br/+ mouse compared with wild-type littermates. The Br/+ mouse also developed a significant increase in blood urea nitrogen at baseline that was further elevated when 2% NaCl was given. Fluid restriction for 48 h further enhanced plasma osmolality and plasma Na(+) responses, although the Br/+ mouse was evidently able to produce a small amount of concentrated urine at this time. Hypothalamic c-Fos expression was appropriately activated in the Br/+ mouse in response to both osmotic challenges, indicating an intact central neuroendocrine pathway that was not affected by the lack of congenital six2 expression. Collectively, our results demonstrate impaired osmoregulatory mechanisms consistent with chronic renal failure in the Br/+ mouse and indicate that six2 haploinsufficiency has a direct effect on postnatal fluid and electrolyte handling associated with fluid imbalance.