Craniofacial and Brain Development

Our research has focused on fundamentally important questions in development

• How does the mouth form?
• What is the Extreme Anterior Domain?
• What is the brain ventricular system?
• How does the brain get its shape?
• How does the brain know where to form?

Embryologists had noted an anterior region where ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. I named the region the ‘Extreme Anterior Domain’ (EAD). We used the Xenopus cement gland as a positional marker for this region, and my group identified the molecular signals that position this organ. My group defined the mouth as an EAD derivative, and identified processes and genes involved in mouth formation. Using a novel facial transplant method we devised, my group showed that the EAD is a facial organizer, which guides neural crest into the developing face. Recent data demonstrates that EAD signaling coordinates brain and face size and may be a novel target for microcephaly. Activity of the EAD is likely to be conserved in mammals.

1. Cement gland-specific activation of the Xag1 promoter is regulated by cooperation of putative Ets and ATF/CREB transcription factors. Wardle F.C., Wainstock D.H., Sive H. 129: 4387-97, 2002.
2. *Jacox, L., *Sindelka, R., Chen, J., Rothman, A., Dickinson, A. and Sive, H. The extreme anterior domain is an essential craniofacial organizer acting through Kinin-Kallikrein signaling. Cell Rep. 8: 596-609, 2014. * equal contribution.
3. Jacox, L., Chen, J., Rothman, A., Lathrop-Marshall, H. and Sive, H. Formation of a ‘pre-mouth  array’ from the extreme anterior domain is directed by neural crest and Wnt/PCP signaling. Cell Rep. 16: 1445-55, 2016.
4. Chen, J. Saldanha, F., Tran, T.H., Vleminckx, K. and Sive, H. Regulation of head size by the Extreme Anterior Domain, a target for microcephaly. iScience, under revision.

The vertebrate nervous system arises from a neural tube, whose cavity of the tube later forms the brain ventricular system. My group pioneered zebrafish as an accessible model for study of brain ventricle development and function. We identified multiple mutants that impact zebrafish brain ventricle formation including the NaK-ATPase. We developed a drainage assay that showed necessity for CSF in brain cell survival. Using mass spectrometry and CSF complementation assays, we identified Retinol Binding Protein 4 (RBP4) as an essential CSF factor. We quantified CSF flow during the major period of neurogenesis, showing possibility for localized CSF control.

1. Lowery, L.A. and Sive, H. Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.  Development, 132: 2057-2067, 2005.
2. Chang, J.T., Lehtinen, M.K. and Sive, H. Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway.  Neurobiol., 76: 75-92, 2016.
3. Fame, R.M., Chang, J.T., Hong, A., Aponte-Santiago, N.A. and Sive, H. Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish. Fluids Barriers CNS 13: 11, 2016. 
4. *Fame, R.M.,*Cortes-Campos, C. and H. Brain Ventricular System and Cerebrospinal Fluid Development and Function: Light at the End of the Tube. BioEssays. 42: doi: 10.1002/bies.201900186, 2020. * equal contribution.

Brain formation not only requires patterning, but also formation of proper 3D shape. We identified and named ‘basal constriction’ as a novel morphogenetic process at the zebrafish midbrain-hindbrain constriction, where epithelial cells narrow adjacent to the basement membrane. Basal constriction likely occurs in every organ as it forms. We identified and named another morphogenetic mechanism that confers ‘stretchiness’ to the neuroepithelium, to allow full expansion of the developing brain. We termed this process ‘epithelial relaxation’.

1. *Gutzman, J.H., *Graeden, E.G., Lowery, L.A., Holley, H.S. and Sive, H. Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction. Mech.Dev. 125: 974-983, 2008.  PMCID: PMC2780020.
2. Gutzman JH, Sive H. Epithelial relaxation mediated by the myosin phosphatase Mypt1 is required for brain ventricle lumen formation and hindbrain morphogenesis. Development. 137:795-804. doi: 10.1242/dev.042705, 2010.
3. *Gutzman J.H., *Graeden E.*, Brachmann I., Yamazoe S., Chen J.K., Sive H.

   Basal constriction during midbrain-hindbrain boundary morphgenesis is mediated by Wnt5b and focal adhesion kinase. Biol Open. 7(11). doi: 10.1242/bio.034520, 2018.

Classical embryological analyses suggested that neural determination and patterning occurred during gastrulation, however no molecular data existed on these key aspects of development. Using a novel subtractive cloning method, we isolated regulatory genes whose expression demonstrated that the nervous system has an anteroposterior axis by early gastrula in both Xenopus and zebrafish. Our work identified retinoic acid as a neural (and mesodermal) posteriorizing factor. We developed the first zebrafish explant system, and were first to use in the embryo dexamethasone-inducible gene expression, now widely employed.

1. Kuo, J., Patel, M., Gamse, J., Merzdorf, C., Liu, X. Apekin, V. and Sive, H. opl: a zinc finger protein that regulates neural determination and patterning in Xenopus. Development 125: 2867-2882, 1998.
2. Sagerström, C., Grinblat, Y. and Sive, H.   Anteroposterior patterning in the zebrafish, Danio: an explant analysis reveals inductive and suppressive cell interactions. Development 122, 1873-1883, 1996.
3. Grinblat, J., #Gamse, J., Patel, M. and Sive, H.  Determination of the zebrafish forebrain: induction and patterning. Development 125: 4403-4416, 1998.
4. Wiellette, E.L. and Sive, H. vhnf1 and FGF signals synergize to specify rhombomere identity in the zebrafish hindbrain.  Development 130: 3821-3829, 2003.