Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in murine cardiopharyngeal mesoderm.

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm within the pharyngeal apparatus. They are disrupted in patients with 22q11.2 deletion syndrome, due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we now identify a multilineage primed population within the cardiopharyngeal mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. The multilineage primed cells are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of cardiopharyngeal mesoderm progenitors. Tbx1 regulates the maturation of multilineage primed progenitor cells to cardiopharyngeal mesoderm derivatives while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance of gene expression by direct and indirect regulation of enriched genes in multilineage primed progenitors and downstream pathways, partly through altering chromatin accessibility, the perturbation of which can lead to congenital defects in individuals with 22q11.2 deletion syndrome.

GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.

Effect of retinoic acid signaling on Ripply3 expression and pharyngeal arch morphogenesis in mouse embryos.

BACKGROUND: Pharyngeal arches (PA) are sequentially generated in an anterior-to-posterior order. Ripply3 is essential for posterior PA development in mouse embryos and its expression is sequentially activated in ectoderm and endoderm prior to formation of each PA. Since the PA phenotype of Ripply3 knockout (KO) mice is similar to that of retinoic acid (RA) signal-deficient embryos, we investigated the relationship between RA signaling and Ripply3 in mouse embryos. RESULTS: In BMS493 (pan-RAR antagonist) treated embryos, which are defective in third and fourth PA development, Ripply3 expression is decreased in the region posterior to PA2 at E9.0. This expression remains and its distribution is expanded posteriorly at E9.5. Conversely, high dose RA exposure does not apparently change its expression at E9.0 and 9.5. Knockout of retinaldehyde dehydrogenase 2 (Raldh2), which causes more severe PA defect, attenuates sequential Ripply3 expression at PA1 and reduces its expression level. EGFP reporter expression driven by a 6 kb Ripply3 promoter fragment recapitulates the endogenous Ripply3 mRNA expression during PA development in wild-type, but its distribution is expanded posteriorly in BMS493-treated and Raldh2 KO embryos. CONCLUSION: Spatio-temporal regulation of Ripply3 expression by RA signaling is indispensable for the posterior PA development in mouse.

Single-cell reconstruction with spatial context of migrating neural crest cells and their microenvironments during vertebrate head and neck formation.

The dynamics of multipotent neural crest cell differentiation and invasion as cells travel throughout the vertebrate embryo remain unclear. Here, we preserve spatial information to derive the transcriptional states of migrating neural crest cells and the cellular landscape of the first four chick cranial to cardiac branchial arches (BA1-4) using label-free, unsorted single-cell RNA sequencing. The faithful capture of branchial arch-specific genes led to identification of novel markers of migrating neural crest cells and 266 invasion genes common to all BA1-4 streams. Perturbation analysis of a small subset of invasion genes and time-lapse imaging identified their functional role to regulate neural crest cell behaviors. Comparison of the neural crest invasion signature to other cell invasion phenomena revealed a shared set of 45 genes, a subset of which showed direct relevance to human neuroblastoma cell lines analyzed after exposure to the in vivo chick embryonic neural crest microenvironment. Our data define an important spatio-temporal reference resource to address patterning of the vertebrate head and neck, and previously unidentified cell invasion genes with the potential for broad impact.

Physiological electric fields induce directional migration of mammalian cranial neural crest cells.

During neurulation, cranial neural crest cells (CNCCs) migrate long distances from the neural tube to their terminal site of differentiation. The pathway traveled by the CNCCs defines the blueprint for craniofacial construction, abnormalities of which contribute to three-quarters of human birth defects. Biophysical cues like naturally occurring electric fields (EFs) have been proposed to be one of the guiding mechanisms for CNCC migration from the neural tube to identified position in the branchial arches. Such endogenous EFs can be mimicked by applied EFs of physiological strength that has been reported to guide the migration of amphibian and avian neural crest cells (NCCs), namely galvanotaxis or electrotaxis. However, the behavior of mammalian NCCs in external EFs has not been reported. We show here that mammalian CNCCs migrate towards the anode in direct current (dc) EFs. Reversal of the field polarity reverses the directedness. The response threshold was below 30 â€‹mV/mm and the migration directedness and displacement speed increased with increase in field strength. Both CNCC line (O9-1) and primary mouse CNCCs show similar galvanotaxis behavior. Our results demonstrate for the first time that the mammalian CNCCs respond to physiological EFs by robust directional migration towards the anode in a voltage-dependent manner.

Surgical and embryological perspective of a big loop of internal carotid artery extending laterally beyond internal jugular vein.

Knowledge of variations of the internal carotid artery is significant to surgeons and radiologists. The internal carotid artery normally runs a straight course in the neck. Its anomalies can lead to its iatrogenic injuries. We report a case of a large loop of the internal carotid artery in a male cadaver aged about 75 years. The common carotid artery terminated by dividing it into the external carotid artery and internal carotid arteries at the level of the upper border of the thyroid cartilage. From the level of origin, the internal carotid artery coursed upwards, backwards and laterally, and formed a large loop behind the internal jugular vein. The variation was found on the left side of the neck and was unilateral. The uncommon looping of the internal carotid artery might result in altered blood flow to the brain and may lead to misperceptions in surgical, imaging, and invasive procedures.

Case report series: revisiting third and fourth pharyngeal arch anomalies, - are they thymopharyngeal duct remnants?

BACKGROUND: Pharyngeal arch anomalies are the second most common form of head and neck congenital defect. The second arch anomalies are the most common, and compromise 95% of cases. Little is known about the 3rd and 4th arch anomalies as they are extremely rare. They most commonly present in childhood with sudden severe left lateral neck infection and abscess formation with considerable tendency to recur, contributing to significant mortality and morbidity in those patients. CASE PRESENTATION: Here we present four cases finally diagnosed as third or fourth pharyngeal arch anomalies, with more than 20 years of follow-up following their definitive surgery. The possibility that they are thymopharyngeal duct remnants is discussed. CONCLUSION: Meticulous open radical surgical excision of all involved paralaryngeal, parapharyngeal and thyroid tissue, with preservation of the superior and recurrent laryngeal nerves, is required for cure of recurrent cases.

Mandibulofacial Dysostosis Attributed to a Recessive Mutation of CYP26C1 in Hereford Cattle.

In spring 2020, six Hereford calves presented with congenital facial deformities attributed to a condition we termed mandibulofacial dysostosis (MD). Affected calves shared hallmark features of a variably shortened and/or asymmetric lower mandible and bilateral skin tags present 2-10 cm caudal to the commissure of the lips. Pedigree analysis revealed a single common ancestor shared by the sire and dam of each affected calf. Whole-genome sequencing (WGS) of 20 animals led to the discovery of a variant (Chr26 g. 14404993T>C) in Exon 3 of CYP26C1 associated with MD. This missense mutation (p.L188P), is located in an α helix of the protein, which the identified amino acid substitution is predicted to break. The implication of this mutation was further validated through genotyping 2 additional affected calves, 760 other Herefords, and by evaluation of available WGS data from over 2500 other individuals. Only the affected individuals were homozygous for the variant and all heterozygotes had at least one pedigree tie to the suspect founder. CYP26C1 plays a vital role in tissue-specific regulation of retinoic acid (RA) during embryonic development. Dysregulation of RA can result in teratogenesis by altering the endothelin-1 signaling pathway affecting the expression of Dlx genes, critical to mandibulofacial development. We postulate that this recessive missense mutation in CYP26C1 impacts the catalytic activity of the encoded enzyme, leading to excess RA resulting in the observed MD phenotype.

J. P. Hill and Katherine Watson's studies of the neural crest in marsupials.

In the early part of the 20th century, J. P. Hill and K. P. Watson embarked on a comprehensive study of the development of the brain in Australian marsupials. Their work included series from three major groups: dasyurids, peramelids, and diprotodonts, covering early primitive streak through brain closure and folding stages. While the major part of the work was on the development of the brain, in the course of this work they documented that cellular proliferations from the neural plate provided much of the mesenchyme of the branchial arches. These proliferations are now known to be the neural crest. However, except for a very brief note, published shortly after Hill's death, this work was never published. In this study, I present Hill and Watson's work on the development of the early neural plate and the neural crest in marsupials. I compare their findings with published work on the South American marsupial, Monodelphis domestica and demonstrate that patterns reported in Monodelphis are general for marsupials. Further, using their data I demonstrate that in dasyurids, which are ultra-altricial at birth, the neural crest migrates early and in massive quantities, even relative to other marsupials. Finally, I discuss the historical context and speculate on reasons for why this work was unpublished. I find little support for ideas that Hill blocked publication because of loyalty to the germ layer theory. Instead, it appears primarily to have been a very large project that was simply orphaned as Watson and Hill pursued other activities.

Cxcr4 and Sdf-1 are critically involved in the formation of facial and non-somitic neck muscles.

The present study shows that the CXCR4/SDF-1 axis regulates the migration of second branchial arch-derived muscles as well as non-somitic neck muscles. Cxcr4 is expressed by skeletal muscle progenitor cells in the second branchial arch (BA2). Muscles derived from the second branchial arch, but not from the first, fail to form in Cxcr4 mutants at embryonic days E13.5 and E14.5. Cxcr4 is also required for the development of non-somitic neck muscles. In Cxcr4 mutants, non-somitic neck muscle development is severely perturbed. In vivo experiments in chicken by means of loss-of-function approach based on the application of beads loaded with the CXCR4 inhibitor AMD3100 into the cranial paraxial mesoderm resulted in decreased expression of Tbx1 in the BA2. Furthermore, disrupting this chemokine signal at a later stage by implanting these beads into the BA2 caused a reduction in MyoR, Myf5 and MyoD expression. In contrast, gain-of-function experiments based on the implantation of SDF-1 beads into BA2 resulted in an attraction of myogenic progenitor cells, which was reflected in an expansion of the expression domain of these myogenic markers towards the SDF-1 source. Thus, Cxcr4 is required for the formation of the BA2 derived muscles and non-somitic neck muscles.

Ontogeny of the digestive tract of Brycon amazonicus (Teleostei, Bryconidae) under culture conditions: from hatching to juvenile stage.

In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.

Pbx4 limits heart size and fosters arch artery formation by partitioning second heart field progenitors and restricting proliferation.

Vertebrate heart development requires the integration of temporally distinct differentiating progenitors. However, few signals are understood that restrict the size of the later-differentiating outflow tract (OFT). We show that improper specification and proliferation of second heart field (SHF) progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts owing to an increase in ventricular and smooth muscle cells. Specifically, Pbx4 initially promotes the partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arches. Subsequently, Pbx4 limits SHF progenitor (SHFP) proliferation. Single cell RNA sequencing of nkx2.5+ cells revealed previously unappreciated distinct differentiation states and progenitor subpopulations that normally reside within the SHF and arterial pole of the heart. Specifically, the transcriptional profiles of Pbx4-deficient nkx2.5+ SHFPs are less distinct and display characteristics of normally discrete proliferative progenitor and anterior, differentiated cardiomyocyte populations. Therefore, our data indicate that the generation of proper OFT size and arch arteries requires Pbx-dependent stratification of unique differentiation states to facilitate both homeotic-like transformations and limit progenitor production within the SHF.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

Zebrafish Pax1a and Pax1b are required for pharyngeal pouch morphogenesis and ceratobranchial cartilage development.

Pharyngeal arches are derived from all three germ layers and molecular interactions among the tissue types are required for proper development of subsequent pharyngeal cartilages; however, the mechanisms underlying this process are not fully described. Here we report that in zebrafish, Pax1a and Pax1b have overlapping and essential functions in pharyngeal pouch morphogenesis and subsequent ceratobranchial cartilage development. Both pax1a and pax1b are co-expressed in pharyngeal pouches, and time-lapse imaging of a novel Tg(pax1b:eGFP) enhancer trap line further revealed the sequential segmental development of pharyngeal pouches. Zebrafish pax1a-/-; pax1b-/- double mutant embryos generated by CRISPR-Cas9 mutagenesis exhibit unsegmented pharyngeal pouches 2-5 with small outpocketings. Endodermal expression of fgf3, tbx1 and edn1 is also absent in pharyngeal pouches 2-5 at 36 h post fertilization (hpf). Loss of ceratobranchial cartilage 1-4 and reduced or absent expression of dlx2a and hand2 in the pharyngeal arches 3-6 are observed in CRISPR mutant and morphant embryos that are deficient in both zebrafish pax1a and pax1b at 96 or 36 hpf. These results suggest that zebrafish Pax1a and Pax1b both regulate pharyngeal pouch morphogenesis by modulating expression of fgf3 and tbx1. Furthermore, our data support a model wherein endodermal Pax1a and Pax1b act through Fgf3 and Tbx-Edn1 signaling to non-autonomously regulate the development of ceratobranchial cartilage via expression of dlx2a and hand2.

Notch signalling regulates epibranchial placode patterning and segregation.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Tbx1 and Foxi3 genetically interact in the pharyngeal pouch endoderm in a mouse model for 22q11.2 deletion syndrome.

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.

Isl-1 positive pharyngeal mesenchyme subpopulation and its role in the separation and remodeling of the aortic sac in embryonic mouse heart.

BACKGROUND: Second heart field cells and neural crest cells have been reported to participate in the morphogenesis of the pharyngeal arch arteries (PAAs); however, how the PAAs grow out and are separated from the aortic sac into left and right sections is unknown. RESULTS: An Isl-1 positive pharyngeal mesenchyme protrusion in the aortic sac ventrally extends and fuses with the aortic sac wall to form a midsagittal septum that divides the aortic sac. The aortic sac division separates the left and right PAAs to form independent arteries. The midsagittal septum dividing the aortic sac has a different expression pattern from the aortic-pulmonary (AP) septum in which Isl-1 positive cells are absent. At 11 days post-conception (dpc) in a mouse embryo, the Isl-1 positive mesenchyme protrusion appears as a heart-shaped structure, in which subpopulations with Isl-1+ Tbx3+ and Isl-1+ Nkx2.5+ cells are included. CONCLUSIONS: The aortic sac is a dynamic structure that is continuously divided during the migration from the pharyngeal mesenchyme to the pericardial cavity. The separation of the aortic sac is not complete until the AP septum divides the aortic sac into the ascending aorta and pulmonary trunk. Moreover, the midsagittal septum and the AP septum are distinct structures.